Novel Class of Therapeutic Protein Based Molecules

ABSTRACT

The present invention provides new compositions and methods for preventing and treating pathogen infection. In particular, the present invention provides compounds having an anchoring domain that anchors the compound to the surface of a target cell, and a therapeutic domain that can act extracellularly to prevent infection of a target cell by a pathogen, such as a virus. The present invention also comprises therapeutic compositions having sialidase activity, including protein-based compounds having sialidase catalytic domains. Compounds of the invention can be used for treating or preventing pathogen infection, and for treating and reducing allergic and inflammatory responses. The invention also provides compositions and methods for enhancing transduction of target cells by recombinant viruses. Such compositions and methods can be used in gene therapy.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.12/861,392, filed Aug. 23, 2010, entitled “A Novel Class of TherapeuticProtein Based Molecules”, which is a continuation of U.S. applicationSer. No. 10/939,262, filed Sep. 10, 2004, entitled “A Novel Class ofTherapeutic Protein Based Molecules”, which claims benefit of priorityto U.S. Provisional Application No. 60/561,749, filed Apr. 13, 2004,entitled “Anti-microbial therapeutics and prophylaxis”, and benefit ofpriority to U.S. Provisional Application No. 60/580,084, filed Jun. 16,2004, entitled “Class of broad spectrum anti-microbial agents.” Theaforementioned applications are all herein incorporated by reference.

BACKGROUND OF THE INVENTION

The invention relates to therapeutic compositions that can be used toprevent and treat infection of human and animal subjects by a pathogen,and specifically to protein-based therapeutic compositions that can beused for the prevention and treatment of viral or bacterial infections.The invention also relates to therapeutic protein-based compositionsthat can be used to prevent or ameliorate allergic and inflammatoryresponses. The invention also relates to protein-based compositions forincreasing transduction efficiency of a recombinant virus, such as arecombinant virus used for gene therapy.

Influenza is a highly infectious acute respiratory disease that hasplagued the human race since ancient times. It is characterized byrecurrent annual epidemics and periodic major worldwide pandemics.Because of the high disease-related morbidity and mortality, direct andindirect social economic impacts of influenza are enormous. Yearlyepidemics cause approximately 300,000 hospitalizations and 25,000 deathsin the United States alone. Four pandemics occurred in the last century;together they caused tens of millions of deaths. Mathematical modelsbased on earlier pandemic experiences have estimated that 89,000-207,000deaths, 18-42 million outpatient visits and 20-47 million additionalillnesses will occur during the next pandemic (Meltzer, M I, Cox, N Jand Fukuda, K. (1999) Emerg Infect Dis 5:659-671).

Influenza is typically caused by infection of two types of viruses,Influenza virus A and Influenza virus B (the third type Influenza virusC only causes minor common cold like symptoms). They belong to theorthomyxoviridae family of RNA viruses. Both type A and type B viruseshave 8 segmented negative-strand RNA genomes enclosed in a lipidenvelope derived from the host cell. The viral envelope is covered withspikes that are composed of three types of proteins: hemagglutinin (HA)which attaches virus to host cell receptors and mediates fusion of viraland cellular membranes; neuraminidase (NA) which facilitates the releaseof the new viruses from host cells; and a small number of M2 proteinswhich serve as ion channels.

Infections by influenza type A and B viruses are typically initiated atthe mucosal surface of the upper respiratory tract. Viral replication isprimarily limited to the upper respiratory tract but can extend to thelower respiratory tract and cause bronchopneumonia that can be fatal.

Influenza viral protein hemagglutinin (HA) is the major viral envelopeprotein. It plays an essential role in viral infection. The importanceof HA is evidenced by the fact that it is the major target forprotective neutralizing antibodies produced by the host immune response(Hayden, F G. (1996) In Antiviral drug resistance (ed. D. D. Richman),pp. 59-77. Chichester, UK: John Wiley & Sons Ltd.). It is now clear thatHA has two different functions in viral infection. First, HA isresponsible for the attachment of the virus to sialic acid cellreceptors. Second, HA mediates viral entry into target cells bytriggering fusion of the viral envelope with cellular membranes.

HA is synthesized as a precursor protein, HA0, which is transferredthrough the Golgi apparatus to the cell surface as a trimeric molecularcomplex. HA0 is further cleaved to generate the C terminus HA1 (residue328 of HA0) and the N terminus of HA2. It is generally believed that thecleavage occurs at the cell surface or on released viruses. The cleavageof HA0 into HA1/HA2 is not required for HA binding to sialic acidreceptor; however, it is believed to be necessary for viral infectivity(Klenk, H D and Rott, R. (1988) Adv Vir Res. 34:247-281; Kido, H, Niwa,Y, Beppu, Y. and Towatari, T. (1996) Advan Enzyme Regul 36:325-347;Skehel, J J and Wiley, D C. (2000) Annu Rev Biochem 69:531-569; Zambon,M. (2001) Rev Med Virol 11:227-241.)

Currently, influenza is controlled by vaccination and anti-viralcompounds. Inactivated influenza vaccines are now in worldwide use,especially in high-risk groups. The vaccine viruses are grown in fertilehen's eggs, inactivated by chemical means and purified. The vaccines areusually trivalent, containing representative influenza A viruses (H1N1and H3N2) and influenza B strains. The vaccine strains need to beregularly updated in order to maintain efficacy; this effort iscoordinated by the World Health Organization (WHO). Duringinter-pandemic periods, it usually takes 8 months before the updatedinfluenza vaccines are ready for the market (Wood, J. (2001) Phil TransR Soc Lond B 356:1953-1960). However, historically, pandemics spread tomost continents within 6 months, and future pandemics are expected tospread even faster with increased international travel (Gust, I D,Hampson, A W., and Lavanchy, D. (2001) Rev Med Virol 11:59-70).Therefore it is inevitable that an effective vaccine will be unavailableor in very short supply during the first waves of future pandemics.

Anti-viral compounds have become the mainstay for treatinginter-pandemic diseases. Currently, they are also the only potentialalternative for controlling pandemics during the initial period whenvaccines are not available. Two classes of antiviral compounds arecurrently on the market: the M2 inhibitors, such as amantadine andrimantadine; and the NA inhibitors, which include oseltamivir (Tamiflu)and zanamivir (Relenza). Both classes of molecules have proven efficacyin prevention and treatment of influenza. However, side effects and therisk of generating drug-resistant viruses remain the top two concernsfor using them widely as chemoprophylaxis (Hayden, F G. (1996) InAntiviral drug resistance (ed. D. D. Richman), pp. 59-77. Chichester,UK: John Wiley & Sons Ltd.). Most importantly, future pandemic strains,either evolved naturally or artificially created by genetic engineeringin bio-warfare, may be resistant to all the available anti-viralcompounds, and this will have devastating consequences globally.

In summary, currently available vaccination and anti-viral compounds arelimited by some fundamental shortcomings. Novel therapeutic andprophylactic modalities are needed to address future influenzapandemics.

Respiratory tract infections (RTIs) are the most common, and potentiallymost severe, types of infectious diseases. Clinically, RTIs includesinusitis, otitis, laryngitis, bronchitis and pneumonia. Based onnumerous etiology and epidemiology studies, it is clear that althoughmany microorganisms have the potential to cause RTIs, only a handful ofpathogens are responsible for vast majority of the cases. Such pathogensinclude S. pneumoniae, M. pneumoniae, H. influenzae, M. catarrhalis,influenza A & B, and parainfluenza virus. Besides causing CAP and AECB,several of the bacterial pathogens, such as S. pneumoniae and H.influenzae, are also the common cause of acute sinusitis, otitis media,as well as invasive infections leading to sepsis, meningitis, etc.Therefore these microorganisms are of the highest clinical importance.

One common feature of all respiratory pathogenic bacteria is that theyestablish commensal colonization on the mucosal surface of the upperairway; such colonization precedes an infection and is prerequisite forinfections. The bacterial colonization in a neonate occurs shortly afterbirth. During lifetime, the upper airway, specifically the nasopharynxand oropharynx, remains a dynamic ecological reservoir of microbialspecies with bacteria being acquired, eliminated and re-acquiredcontinually. In most cases the bacterial flora in the pharynx isharmless. However, when the condition of the host is altered, somemicroorganisms may invade adjacent tissues or bloodstream to causediseases. In addition to serving as the port of entry for mucosal andinvasive infections by both bacteria and viruses, the nasopharynx isalso the major source of spreading the pathogenic microorganisms betweenindividuals, as well as the reservoir where antibiotic-resistantbacteria are selected (Garcia-Rodriguez and Martinez, J AntimicrobChemother, (2002) 50 (Suppl S2), 59-73; Soriano and Rodriguez-Cerrato, JAntimicrob Chemother, (2002) 50 (Suppl S2), 51-58). It is wellestablished clinically that individuals who are prone to RTIs tend to bepersistent and recurrent carriers of the pathogenic bacteria(Garcia-Rodriguez and Martinez, J Antimicrob Chemother, (2002) 50 (SupplS2), 59-73; Mbaki et al., Tohoku J. Exp. Med., (1987) 153 (2), 111-121).

Helicobacter pylori is a human pathogen implicated in gastritis andpeptic ulcer. The bacterium resides in the human stomach and binds toepithelial cells of the gastric antrum. It has been demonstrated thatthe bacterial adhesion is mediated by binding of Helicobacter pyloriadhesin I and II to sialic acids on the epithelial surface.

Siglecs (sialic acid binding Ig-like lectins) are members of theimmunoglobulin (Ig) superfamily that bind to sialic acid and are mainlyexpressed by cells of the hematopoietic system. At least 11 siglecs havebeen discovered and they seem to exclusively recognize cell surfacesialic acid as the ligand. It is believed that the binding of siglecs tosialic acid mediates cell-cell adhesion and interactions (Crocker andVarki, Trends Immunol., (2001) 22 (6), 337-342; Angata and Brinkman-Vander Linden, Biochim. Biophys. Acta, (2002) 1572 (2-3), 294-316).Siglec-8 (SAF-2) is an adhesion molecule that is highly restricted tothe surface of eosinophils, basophils, and mast cells, which are thecentral effector cells in allergic conditions including allergicrhinitis, asthma and eczema. Siglec-8 is considered to be responsiblefor mediating the recruitment of the three allergic cell types to theairway, the lungs and other sites of allergy. Siglec-1 (sialoadhesion)and siglec-2 (CD22) are the adhesion molecules on macrophages and Bcells, both types of cells play central roles in immune reactions thatlead to inflammation.

Recombinant viruses, in particular adeno-associated virus (AAV), can beused to transfer the wild type cystic fibrosis transmembrane conductanceregulator (CFTR) gene into the epithelial cells to correct the geneticdefect that causes cystic fibrosis (Flotte and Carter, Methods Enzymol.,(1998) 292, 717-732). Clinical trials with AAV vectors have shownefficient and safe delivery of the CFTR gene into epithelial cells withlow levels of gene transfer (Wagner et al., Lancet, (1998) 351 (9117),1702-1703). Compared to adenoviral vectors, AAV offers more stable geneexpression and diminished cellular immunity. However, the transductionefficiency of AAV in vivo is rather low in the lung (Wagner et al.,Lancet, (1998) 351 (9117), 1702-1703). A method that can improvetransduction efficiency of AAV in vivo is needed to achieve fulltherapeutic potential of gene therapy for cystic fibrosis. It has beenshown that negatively charged carbohydrates, such as sialic acid,inhibit the transduction efficiency of AAV vector to thewell-differentiated airway epithelium, and treatment of the airwayepithelium by glycosidases, including a neuraminidase, andendoglycosidase H, enhances transduction efficiency of the AAV vector(Bals et al., J. Virol., (1999) 73 (7), 6085-6088).

BRIEF SUMMARY OF THE INVENTION

The present invention recognizes that current therapeutics forpreventing and treating infection by pathogens are often difficult toprovide in a timely manner, can have undesirable side effects, and canlead to drug-resistant pathogen strains. The present invention alsorecognizes that the current approach to treat allergy and inflammationhas limited efficacy and is associated with side effects. In addition,the present invention also recognizes that the current approach toadminister recombinant viruses yield low transduction efficiency andunsatisfactory efficacy of the gene therapy.

The present invention provides new compositions and methods forpreventing and treating pathogen infection. In particular, the presentinvention provides compounds that can act extracellularly to preventinfection of a cell by a pathogen. Some preferred embodiments of thepresent invention are therapeutic compounds having an anchoring domainthat anchors the compound to the surface of a target cell, and atherapeutic domain that can act extracellularly to prevent infection ofthe target cell by a pathogen, such as a virus or bacterium.

In one aspect, the invention provides a protein-based composition forpreventing or treating infection by a pathogen. The compositioncomprises a compound that comprises at least one therapeutic domaincomprising a peptide or protein, where the therapeutic domain has atleast one extracellular activity that can prevent the infection of atarget cell by a pathogen, and at least one anchoring domain that canbind at or near the membrane of a target cell.

In some embodiments of this aspect of the present invention, the atleast one therapeutic domain comprises an inhibitory activity thatprevents or impedes the infection of a target cell by a pathogen. In apreferred embodiment, the inhibitory activity inhibits the activity of aprotease that can process a viral protein necessary for infection of atarget cell. In a particularly preferred embodiment, the compoundcomprises a therapeutic domain that can inhibit the processing of the HAprotein of influenza virus, and the anchoring domain can bind thecompound at the surface of a respiratory epithelial cell.

In some embodiments of the present invention, at least one therapeuticdomain comprises a catalytic activity. In a preferred embodiment, thecatalytic activity removes a moiety from the surface of a target cellthat is necessary for infection of the target cell. In a particularlypreferred embodiment, the therapeutic domain is a sialidase that candigest sialic acid moieties on the surface of epithelial target cells,and the anchoring domain is a GAG-binding domain of a human protein thatcan bind heparin or heparan sulfate moieties at the surface of anepithelial cell.

In another aspect, the present invention includes pharmaceuticalcompositions for treating or preventing pathogen infection in a subject.Pharmaceutical compositions comprise a compound of the present inventioncomprising at least one therapeutic domain and at least one anchoringdomain. The pharmaceutical composition can also comprise solutions,stabilizers, fillers and the like. In some preferred embodiments, thepharmaceutical composition is formulated as an inhalant. In somepreferred embodiments, the pharmaceutical composition is formulated as anasal spray.

Another aspect of the present invention is a pharmaceutical compositioncomprising at least one sialidase. The sialidase can be isolated fromany source, such as, for example, a bacterial or mammalian source, orcan be a recombinant protein that is substantially homologous to anaturally occurring sialidase. A pharmaceutical composition comprising asialidase can be formulated for nasal, tracheal, bronchial, oral, ortopical administration, or can be formulated as an injectable solutionor as eyedrops. A pharmaceutical composition comprising a sialidase canbe used to treat or prevent pathogen infection, to treat or preventallergy or inflammatory response, or to enhance the transductionefficiency of a recombinant virus for gene therapy. Yet another aspectof the present invention is a sialidase catalytic domain protein.

In this aspect, proteins that comprise the catalytic domain of asialidase but comprise less than the entire sialidase the catalyticdomain sequence is derived from are considered sialidase catalyticdomain proteins. Sialidase catalytic domain proteins can comprise otherprotein sequences, such as but not limited to functional domains derivedfrom other proteins. A pharmaceutical composition comprising a sialidasecan be formulated for nasal, tracheal, bronchial, oral, or topicaladministration, or can be formulated as an injectable solution or aseyedrops. A pharmaceutical composition comprising a sialidase can beused to treat or prevent pathogen infection, to treat or prevent allergyor inflammatory response, or to enhance the transduction efficiency of arecombinant virus for gene therapy.

In yet another aspect, the present invention includes a method fortreating or preventing infection by a pathogen. In preferredembodiments, the method comprises administering a sialidase activity,such as a sialidase or a sialidase catalytic domain protein, including asialidase catalytic domain fusion protein, to a subject to prevent ortreat an infection. A pathogen can be, for example, a viral or bacterialpathogen. The method includes applying a pharmaceutically effectiveamount of a compound of the present invention to at least one targetcell of a subject. Preferably, the pharmaceutical composition canapplied by the use of a spray, inhalant, or topical formulation.

The present invention also provides new compositions and methods fortreating allergy and inflammation. In particular, the present inventionprovides compounds that can act extracellularly to prevent or inhibitadhesion and function of inflammatory cells. Some preferred embodimentsof compounds for treating allergy or inflammation comprise at least onetherapeutic domain that has the said extracellular activity and an atleast one anchoring domain that anchors the compound to the surface of atarget cell. In some preferred embodiments, the method comprisesadministering a siaidase activity, such as a sialidase or a sialidasecatalytic domain protein, including a sialidase catalytic domain fusionprotein to a subject to prevent or treat an allergic or inflammatoryresponse. The allergic or inflammatory response can be asthma, allergicrhinitis, skin conditions such as eczema, or response to plant or animaltoxins. The method includes applying a pharmaceutically effective amountof a compound of the present invention to at least one target cell of asubject. Preferably, the pharmaceutical composition can applied by theuse of a spray, inhalant, or topical formulation.

The present invention also provides new compositions and methods forimproving efficiency of gene transfer by recombinant viral vectorsduring gene therapy. In particular, the present invention providescompounds that can act extracellularly to reduce the physical orchemical barrier that hinders transduction by gene therapy vectors, suchas AAV vector. Some preferred compounds of the present invention forimproving efficiency of gene transfer by recombinant viral vectorscomprise at least one therapeutic domain that has an extracellularactivity and an at least one anchoring domain that anchors the compoundto the surface of a target cell. In some preferred embodiments, themethod comprises administering a siaidase activity, such as a sialidaseor a sialidase catalytic domain protein, including a sialidase catalyticdomain fusion protein to a subject to facilitate transduction of atarget cell by a recombinant viral vector. The method includes applyingan effective amount of a compound of the present invention along with arecombinant viral vector to at least one target cell. A pharmaceuticalcomposition of the present invention can be applied by the use of aspray, inhalant, or topical formulation.

BRIEF DESCRIPTION OF SEVERAL VIEWS OF THE DRAWINGS

FIG. 1 is a schematic depiction of the primary amino acid structure ofaprotinin.

FIG. 2 shows GAG-binding sequences of six human genes: PF4, humanplatelet factor 4; IL8, human interleukin 8; AT III, human antithrombinIII; ApoE, human apolipoprotein E; AAMP, human angio-associatedmigratory cell protein; AR, human amphiregulin.

FIG. 3 is a sequence comparison between human sialidases NEU2 and NEU4.

FIG. 4 is a table comparing substrate specificity of bacterial andfungal sialidases.

FIG. 5 depicts the nucleotide (SEQ ID NO:28) and amino acid (SEQ IDNO:29) sequences of Construct #1 encoding His6-AvCD. NcoI and HindIIIsites used for cloning into pTrc99a are shown in bold.

FIG. 6 depicts the nucleotide (SEQ ID NO:18) and amino acid (SEQ IDNO:19) sequences of Construct #2 encoding AR-AvCD. NcoI and HindIIIsites used for cloning into pTrc99a are shown in bold.

FIG. 7 depicts the nucleotide (SEQ ID NO:36) and amino acid (SEQ IDNO:37) sequences of Construct #3 encoding AR-G₄S-AvCD. NcoI and HindIIIsites used for cloning into pTrc99a are shown in bold.

FIG. 8 is a graph of data from an experiment showing that the AR-tagenhances the removal of α(2,6)-linked sialic acid from MDCK cells. The Yaxis shows the percentage of α(2,6)-linked sialic acid remaining on thesurface of MDCK cells after treatment with various dilutions ofrecombinant AvCD (Construct #1) (diamonds) or recombinant AR-AvCD(Construct #2) (squares).

FIG. 9 is a graph depicting the protection against influenza virusesconferred by treating MDCK cells with recombinant AR-AvCD protein madefrom Construct #2 or the isolated sialidase of A. ureafaciens. Thechallenge viral strains are: A/WS/33 (H1N1); A/PR/8 (H1N1);A/Japan/305/57 (H2N2); A/Victoria/504/2000 (H3N2); A/HongKong/8/68(H3N2); B/Lee/40; 7. B/Maryland/1/59; and Turkey/W is/66 (H9N2).

FIG. 10 is a graph showing the level of inhibition of influenza virusamplification by the recombinant AR-AvCD sialidase and the recombinantAR-G₄S-AvCD sialidase. The challenge viral strains are: A/PR/8 (H1N1);A/WS/33 (H1N1); A/Japan/305/57 (H2N2); A/HongKong/8/68 (H3N2); B/Lee/40;7. B/Maryland/1/59; and Turkey/W is/66 (H9N2).

FIG. 11 provides graphs showing that topical administration ofrecombinant AR-AvCD sialidase fusion protein reduces the inflammatoryresponses of ferrets infected with an influenza A (H1N1) virus. (A) Thetotal number of inflammatory cells from nasal wash samples obtained frominfected animals at the indicated times after infection. (B) The proteinconcentration was determined in cell-free nasal wash samples of infectedferrets. Infected ferrets were vehicle-treated (squares) or were treatedwith recombinant AR-AvCD sialidase fusion protein made from Construct #2(triangles). Uninfected animals were also treated with recombinantAR-AvCD sialidase fusion protein (diamonds). Statistically significantvalues are labeled with * (p<0.05) and ** (p<0.01).

FIG. 12 is a table depicting inhibition of viral replication, cellprotection EC50's, and selective indexes for two sialidase catalyticdomain fusion proteins of the present invention. All EC50's are inmU/ml.

FIG. 13 is a table depicting viral replication in the respiratory tractof ferrets treated with a sialidase catalytic domain fusion proteins ofthe present invention and ferrets treated with a control vehicle.

DETAILED DESCRIPTION OF THE INVENTION Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Generally, the nomenclatureused herein and the manufacture or laboratory procedures described beloware well known and commonly employed in the art. Conventional methodsare used for these procedures, such as those provided in the art andvarious general references. Where a term is provided in the singular,the inventors also contemplate the plural of that term. Where there arediscrepancies in terms and definitions used in references that areincorporated by reference, the terms used in this application shall havethe definitions given herein. As employed throughout the disclosure, thefollowing terms, unless otherwise indicated, shall be understood to havethe following meanings:

A “pathogen” can be any virus or microorganism that can infect a cell, atissue or an organism. A pathogen can be a virus, bacterium, orprotozoan.

A “target cell” is any cell that can be infected by a pathogen or anycell that can interact with inflammatory cells, or a host cell that isthe intended destination for an exogenous gene transferred by arecombinant virus.

A “recombinant virus” or a “recombinant viral vector”, a “gene therapyviral vector” or a “gene therapy vector” is defined as a geneticallyengineered virus that comprises one or more exogenous genes. When atarget cell is transduced by a recombinant virus, the exogenous gene(s)is transferred to the target cell. Genes transferred to a target cellcan be expressed in the cell to provide the intended therapeuticeffects. Currently, most commonly used gene therapy viral vectors arebased on four types of viruses: retrovirus (including lentivirus),adenovirus, adeno-associated virus (AAV) and herpes simplex virus type1.

“Inflammatory cells” are the cells that carry out or participate ininflammatory responses of the immune system. Inflammatory cells includeB lymphocytes, T lymphocytes, macrophages, basophils, eosinophils, mastcells, NK cells and monocytes.

An “extracellular activity that can prevent the infection of a targetcell by a pathogen” is any activity that can block or impede infectionof a target cell by a pathogen by acting at or near the exterior surfaceof a target cell. An extracellular activity that can prevent theinfection of a target cell by a pathogen, can be an activity such as,but not limited to, a catalytic activity or an inhibitory activity. Forexample, a catalytic activity can be an enzymatic activity that degradesone or more entities (such as but not limited to ligands, receptors, orenzymes) on a pathogen, on a target cell, or in the vicinity of a targetcell, in which the one or more entities contribute to the infectionprocess. A catalytic activity can also modify one or more entities on apathogen, on a target cell, or in the vicinity of a target cell, suchthat the infection-promoting property of the entity is reduced. Aninhibitory activity can be an activity that, for example, binds to areceptor or ligand and prevents the receptor or ligand from binding amoiety, where the binding is necessary for or promotes the infectionprocess. An inhibitory activity can also be an inhibitor of an enzyme orreceptor that prevents the enzyme or receptor from performing a functionthat is necessary for or promotes the infection process. The exterior ofa target cell includes the target cell membrane itself, as well as theextracellular milieu surrounding the target cell, includingextracellular matrix, intracellular spaces, and luminal spaces. Forepithelial cells, the exterior of a target cell also includes the apicalor luminal surface of the cell membrane that form luminal linings, andthe extracellular milieu near the luminal surface. An “extracellularactivity that can prevent the infection of a target cell by a pathogen”can be any type of chemical entity, including a protein, polypeptide,peptide, nucleic acid, peptide nucleic acid, nucleic acid analogue,nucleotide, nucleotide analogue, small organic molecule, polymer,lipids, steroid, fatty acid, carbohydrate, and the like, includingcombinations of any of these. Preferably, however, the activitycomprises a peptide or protein or coupled to a peptide or protein.

An “extracellular activity that can improve transduction efficiency, orgene transfer efficiency, by a recombinant virus” is any activity thatreduces or eliminates physical or chemical barriers that impedes hostcell entry by a recombinant virus by acting at or near the exteriorsurface of a target cell. An extracellular activity that can improvetransduction efficiency, or gene transfer efficiency, by a recombinantvirus can be an activity such as, but not limited to, a catalyticactivity or an inhibitory activity. For example, a catalytic activitycan be an enzymatic activity that degrades one or more entities (such asbut not limited to ligands, receptors, or enzymes) on a pathogen, on atarget cell, or in the vicinity of a target cell, in which the one ormore entities contribute to the infection process. A catalytic activitycan also modify one or more entities on a pathogen, on a target cell, orin the vicinity of a target cell, such that the infection-promotingproperty of the entity is reduced. An inhibitory activity can be anactivity that, for example, binds to a receptor or ligand and preventsthe receptor or ligand from binding a moiety, where the binding isnecessary for or promotes the infection process. An inhibitory activitycan also be an inhibitor of an enzyme or receptor that prevents theenzyme or receptor from performing a function that is necessary for orpromotes the infection process. The exterior of a target cell includesthe target cell membrane itself, as well as the extracellular milieusurrounding the target cell, including extracellular matrix,intracellular spaces, and luminal spaces. For epithelial cells, theexterior of a target cell also includes the apical or luminal surface ofthe cell membrane that form luminal linings, and the extracellularmilieu near the luminal surface. An “extracellular activity that canprevent the infection of a target cell by a pathogen” can be any type ofchemical entity, including a protein, polypeptide, peptide, nucleicacid, peptide nucleic acid, nucleic acid analogue, nucleotide,nucleotide analogue, small organic molecule, polymer, lipids, steroid,fatty acid, carbohydrate, and the like, including combinations of any ofthese. Preferably, however, the activity comprises a peptide or proteinor coupled to a peptide or protein.

An “extracellular activity that can inhibit adhesion or function ofinflammatory cells” is any activity that can prevent inflammatory cellsfrom contacting the target cell and affecting the normal physiologicalstatus of the target cell.

A “domain that can anchor said at least one therapeutic domain to themembrane of a target cell”, also called an “extracellular anchoringdomain” or simply, “anchoring domain” refers to a chemical entity canthat can stably bind a moiety that is at or on the exterior of a cellsurface or is in close proximity to the surface of a cell. Anextracellular anchoring domain can be reversibly or irreversibly linkedto one or more moieties, such as, preferably, one or more therapeuticdomains, and thereby cause the one or more attached therapeutic moietiesto be retained at or in close proximity to the exterior surface of aeukaryotic cell. Preferably, an extracellular anchoring domain binds atleast one molecule on the surface of a target cell or at least onemolecule found in close association with the surface of a target cell.For example, an extracellular anchoring domain can bind a moleculecovalently or noncovalently associated with the cell membrane of atarget cell, or can bind a molecule present in the extracellular matrixsurrounding a target cell. An extracellular anchoring domain preferablyis a peptide, polypeptide, or protein, and can also comprise anyadditional type of chemical entity, including one or more additionalproteins, polypeptides, or peptides, a nucleic acid, peptide nucleicacid, nucleic acid analogue, nucleotide, nucleotide analogue, smallorganic molecule, polymer, lipids, steroid, fatty acid, carbohydrate, ora combination of any of these.

As used herein, a protein or peptide sequences is “substantiallyhomologous” to a reference sequence when it is either identical to areference sequence, or comprises one or more amino acid deletions, oneor more additional amino acids, or more one or more conservative aminoacid substitutions, and retains the same or essentially the sameactivity as the reference sequence. Conservative substitutions may bedefined as exchanges within one of the following five groups:

-   -   I. Small, aliphatic, nonpolar or slightly polar residues: Ala,        Ser, Thr, Pro, Gly    -   II. Polar, negatively charged residues and their amides: Asp,        Asn, Glu, Gln    -   III. Polar, positively charged residues: His, Arg, Lys    -   IV. Large, aliphatic nonpolar residues: Met, Leu, Ile, Val, Cys    -   V. Large aromatic residues: Phe, Try, Trp        Within the foregoing groups, the following substitution are        considered to be “highly conservative”: Asp/Glu, His/Arg/Lys,        Phe/Tyr/Trp, and Met/Leu/Ile/Val. Semi-conservative        substitutions are defined to be exchanges between two of groups        (I)-(IV) above which are limited to supergroup (A), comprising        (I), (II), and (III) above, or to supergroup (B),        comprising (IV) and (V) above. In addition, where hydrophobic        amino acids are specified in the application, they refer to the        amino acids Ala, Gly, Pro, Met, Leu, Ile, Val, Cys, Phe, and        Trp, whereas hydrophilic amino acids refer to Ser, Thr, Asp,        Asn, Glu, Gln, His, Arg, Lys, and Tyr.

A “sialidase” is an enzyme that can remove a sialic acid residue from asubstrate molecule. The sialidases (N-acylneuraminosylglycohydrolases,EC 3.2.1.18) are a group of enzymes that hydrolytically remove sialicacid residues from sialo-glycoconjugates. Sialic acids are alpha-ketoacids with 9-carbon backbones that are usually found at the outermostpositions of the oligosaccharide chains that are attached toglycoproteins and glycolipids. One of the major types of sialic acids isN-acetylneuraminic acid (Neu5Ac), which is the biosynthetic precursorfor most of the other types. The substrate molecule can be, asnonlimiting examples, an oligosaccharide, a polysaccharide, aglycoprotein, a ganglioside, or a synthetic molecule. For example, asialidase can cleave bonds having alpha(2,3)-Gal, alpha(2,6)-Gal, oralpha(2,8)-Gal linkages between a sialic acid residue and the remainderof a substrate molecule. A sialidase can also cleave any or all of thelinkages between the sialic acid residue and the remainder of thesubstrate molecule. Two major linkages between Neu5Ac and thepenultimate galactose residues of carbohydrate side chains are found innature, Neu5Ac alpha (2,3)-Gal and Neu5Ac alpha (2,6)-Gal. Both Neu5Acalpha (2,3)-Gal and Neu5Ac alpha (2,6)-Gal molecules can be recognizedby influenza viruses as the receptor, although human viruses seem toprefer Neu5Ac alpha (2,6)-Gal, avian and equine viruses predominantlyrecognize Neu5Ac alpha (2,3)-Gal. A sialidase can be anaturally-occurring sialidase, an engineered sialidase (such as, but notlimited to a sialidase whose amino acid sequence is based on thesequence of a naturally-occurring sialidase, including a sequence thatis substantially homologous to the sequence of a naturally-occurringsialidase). As used herein, “sialidase” can also mean the active portionof a naturally-occurring sialidase, or a peptide or protein thatcomprises sequences based on the active portion of a naturally-occurringsialidase.

A “fusion protein” is a protein comprising amino acid sequences from atleast two different sources. A fusion protein can comprise amino acidsequence that is derived from a naturally occurring protein or issubstantially homologous to all or a portion of a naturally occurringprotein, and in addition can comprise from one to a very large number ofamino acids that are derived from or substantially homologous to all ora portion of a different naturally occurring protein. In thealternative, a fusion protein can comprise amino acid sequence that isderived from a naturally occurring protein or is substantiallyhomologous to all or a portion of a naturally occurring protein, and inaddition can comprise from one to a very large number of amino acidsthat are synthetic sequences.

A “sialidase catalytic domain protein” is a protein that comprises thecatalytic domain of a sialidase, or an amino acid sequence that issubstantially homologous to the catalytic domain of a sialidase, butdoes not comprises the entire amino acid sequence of the sialidase thecatalytic domain is derived from, wherein the sialidase catalytic domainprotein retains substantially the same activity as the intact sialidasethe catalytic domain is derived from. A sialidase catalytic domainprotein can comprise amino acid sequences that are not derived from asialidase, but this is not required. A sialidase catalytic domainprotein can comprise amino acid sequences that are derived from orsubstantially homologous to amino acid sequences of one or more otherknown proteins, or can comprise one or more amino acids that are notderived from or substantially homologous to amino acid sequences ofother known proteins.

I. Composition for Preventing or Treating Infection by a Pathogen

The present invention includes peptide or protein-based compounds thatcomprise at least one domain that can anchor at least one therapeuticdomain to the membrane of a eukaryotic cell and at least one therapeuticdomain having an extracellular activity that can prevent the infectionof a cell by a pathogen. By “peptide or protein-based” compounds, it ismeant that the two major domains of the compound have an amino acidframework, in which the amino acids are joined by peptide bonds. Apeptide or protein-based compound can also have other chemical compoundsor groups attached to the amino acid framework or backbone, includingmoieties that contribute to the anchoring activity of the anchoringdomain, or moieties that contribute to the infection-preventing activityor the therapeutic domain. For example, the protein-based therapeuticsof the present invention can comprise compounds and molecules such asbut not limited to: carbohydrates, fatty acids, lipids, steroids,nucleotides, nucleotide analogues, nucleic acid molecules, nucleic acidanalogues, peptide nucleic acid molecules, small organic molecules, oreven polymers. The protein-based therapeutics of the present inventioncan also comprise modified or non-naturally occurring amino acids.Non-amino acid portions of the compounds can serve any purpose,including but not limited to: facilitating the purification of thecompound, improving the solubility or distribution or the compound (suchas in a therapeutic formulation), linking domains of the compound orlinking chemical moieties to the compound, contributing to thetwo-dimensional or three-dimensional structure of the compound,increasing the overall size of the compound, increasing the stability ofthe compound, and contributing to the anchoring activity or therapeuticactivity of the compound.

The peptide or protein-based compounds of the present invention can alsoinclude protein or peptide sequences in addition to those that compriseanchoring domains or therapeutic domains. The additional proteinsequences can serve any purpose, including but not limited to any of thepurposes outlined above (facilitating the purification of the compound,improving the solubility or distribution or the compound, linkingdomains of the compound or linking chemical moieties to the compound,contributing to the two-dimensional or three-dimensional structure ofthe compound, increasing the overall size of the compound, increasingthe stability of the compound, or contributing to the anchoring activityor therapeutic activity of the compound). Preferably any additionalprotein or amino acid sequences are part of a single polypeptide orprotein chain that includes the anchoring domain or domains andtherapeutic domain or domains, but any feasible arrangement of proteinsequences is within the scope of the present invention.

The anchoring domain and therapeutic domain can be arranged in anyappropriate way that allows the compound to bind at or near a targetcell membrane such that the therapeutic domain can exhibit anextracellular activity that prevents or impedes infection of the targetcell by a pathogen. The compound will preferably have at least oneprotein or peptide-based anchoring domain and at least one peptide orprotein-based therapeutic domain. In this case, the domains can bearranged linearly along the peptide backbone in any order. The anchoringdomain can be N-terminal to the therapeutic domain, or can be C-terminalto the therapeutic domain. It is also possible to have one or moretherapeutic domains flanked by at least one anchoring domain on eachend. Alternatively, one or more anchoring domains can be flanked by atleast one therapeutic domain on each end. Chemical, or preferably,peptide, linkers can optionally be used to join some or all of thedomains of a compound.

It is also possible to have the domains in a nonlinear, branchedarrangement. For example, the therapeutic domain can be attached to aderivatized side chain of an amino acid that is part of a polypeptidechain that also includes, or is linked to, the anchoring domain.

A compound of the present invention can have more than one anchoringdomain. In cases in which a compound has more than one anchoring domain,the anchoring domains can be the same or different. A compound of thepresent invention can have more than one therapeutic domain. In cases inwhich a compound has more than one therapeutic domain, the therapeuticdomains can be the same or different. Where a compound comprisesmultiple anchoring domains, the anchoring domains can be arranged intandem (with or without linkers) or on alternate sides of other domains,such as therapeutic domains. Where a compound comprises multipletherapeutic domains, the therapeutic domains can be arranged in tandem(with or without linkers) or on alternate sides of other domains, suchas, but not limited to, anchoring domains.

A peptide or protein-based compound of the present invention can be madeby any appropriate way, including purifying naturally occurringproteins, optionally proteolytically cleaving the proteins to obtain thedesired functional domains, and conjugating the functional domains toother functional domains. Peptides can also be chemically synthesized,and optionally chemically conjugated to other peptides or chemicalmoieties. Preferably, however, a peptide or protein-based compound ofthe present invention is made by engineering a nucleic acid construct toencode at least one anchoring domain and at least one therapeutic domaintogether (with or without nucleic acid linkers) in a continuouspolypeptide. The nucleic acid constructs, preferably having appropriateexpression sequences, can be transfected into prokaryotic or eukaryoticcells, and the therapeutic protein-based compound can be expressed bythe cells and purified. Any desired chemical moieties can optionally beconjugated to the peptide or protein-based compound after purification.In some cases, cell lines can be chosen for expressing the protein-basedtherapeutic for their ability to perform desirable post-translationalmodifications (such as, but not limited to glycosylation).

A great variety of constructs can be designed and their protein productstested for desirable activities (such as, for example, binding activityof an anchoring domain, or a binding, catalytic, or inhibitory activityof a therapeutic domain). The protein products of nucleic acidconstructs can also be tested for their efficacy in preventing orimpeding infection of a target cell by a pathogen. In vitro and in vivotests for the infectivity of pathogens are known in the art, such asthose described in the Examples for the infectivity of influenza virus.

Anchoring Domain

As used herein, an “extracellular anchoring domain” or “anchoringdomain” is any moiety that can stably bind an entity that is at or onthe exterior surface of a target cell or is in close proximity to theexterior surface of a target cell. An anchoring domain serves to retaina compound of the present invention at or near the external surface of atarget cell.

An extracellular anchoring domain preferably binds 1) a moleculeexpressed on the surface of a target cell, or a moiety, domain, orepitope of a molecule expressed on the surface of a target cell, 2) achemical entity attached to a molecule expressed on the surface of atarget cell, or 3) a molecule of the extracellular matrix surrounding atarget cell.

An anchoring domain is preferably a peptide or protein domain (includinga modified or derivatized peptide or protein domain), or comprises amoiety coupled to a peptide or protein. A moiety coupled to a peptide orprotein can be any type of molecule that can contribute to the bindingof the anchoring domain to an entity at or near the target cell surface,and is preferably an organic molecule, such as, for example, nucleicacid, peptide nucleic acid, nucleic acid analogue, nucleotide,nucleotide analogue, small organic molecule, polymer, lipids, steroid,fatty acid, carbohydrate, or any combination of any of these.

A molecule, complex, domain, or epitope that is bound by an anchoringdomain may or may not be specific for the target cell. For example, ananchoring domain may bind an epitope present on molecules on or in closeproximity to the target cell and that occur at sites other than thevicinity of the target cell as well. In many cases, however, localizeddelivery of a therapeutic compound of the present invention willrestrict its occurrence primarily to the surface of target cells. Inother cases, a molecule, complex, moiety, domain, or epitope bound by ananchoring domain may be specific to a target tissue or target cell type.

Target tissue or target cell type includes the sites in an animal orhuman body where a pathogen invades or amplifies. For example, a targetcell can be an endothelial cell that can be infected by a pathogen. Acomposition of the present invention can comprise an anchoring domainthat can bind a cell surface epitope, for example, that is specific forthe endothelial cell type. In another example, a target cell can be anepithelial cell and a composition of the present invention can bind anepitope present on the cell surface of many epithelial cell types, orpresent in the extracellular matrix of different types of epithelialcells. In this case localized delivery of the composition can restrictits localization to the site of the epithelial cells that are targets ofthe pathogen.

A compound for preventing or treating infection by a pathogen cancomprise an anchoring domain that can bind at or near the surface ofepithelial cells. For example, heparan sulfate, closely related toheparin, is a type of glycosaminoglycan (GAG) that is ubiquitouslypresent on cell membranes, including the surface of respiratoryepithelium. Many proteins specifically bind to heparin/heparan sulfate,and the GAG-binding sequences in these proteins have been identified(Meyer, F A, King, M and Gelman, R A. (1975) Biochimica et BiophysicaActa 392: 223-232; Schauer, S. ed., pp 233. Sialic Acids Chemistry,Metabolism and Function. Springer-Verlag, 1982). For example, theGAG-binding sequences of human platelet factor 4 (PF4) (SEQ ID NO:2),human interleukin 8 (IL8) (SEQ ID NO:3), human antithrombin III (AT III)(SEQ ID NO:4), human apoprotein E (ApoE) (SEQ ID NO:5), humanangio-associated migratory cell protein (AAMP) (SEQ ID NO:6), or humanamphiregulin (SEQ ID NO:7) (FIG. 2) have been shown to have very highaffinity (in the nanomolar range) towards heparin (Lee, M K and Lander,A D. (1991) Pro Natl Acad Sci USA 88:2768-2772; Goger, B, Halden, Y,Rek, A, Mosl, R, Pye, D. Gallagher, J and Kungl, A J. (2002) Biochem.41:1640-1646; Witt, D P and Lander A D (1994) Curr Bio 4:394-400;Weisgraber, K H, Rall, S C, Mahley, R W, Milne, R W and Marcel, Y.(1986) J Bio Chem 261:2068-2076). The GAG-binding sequences of theseproteins are distinct from their receptor-binding sequences, so theywill not induce the biological activities associated with thefull-length proteins or the receptor-binding domains. These sequences,or other sequences that have been identified or are identified in thefuture as heparin/heparan sulfate binding sequences, or sequencessubstantially homologous to identified heparin/heparan sulfate bindingsequences that have heparin/heparan sulfate binding activity, can beused as epithelium-anchoring-domains in compounds of the presentinvention that can be used to prevent or treat, for example, respiratoryepithelium-infecting viruses such as, but not limited to, influenzavirus.

An anchoring domain can bind a moiety that is specific to the targetcell type of a particular species or can bind a moiety that is found inthe target cell type of more than one species. In cases where theanchoring domain can bind moieties that are present at the surface oftarget cells of more than one species, and a virus or pathogen caninfect more than one species, a therapeutic compound can have utilityfor more than one species (providing that the therapeutic domain is alsoeffective across the relevant species.) For example, in the case oftherapeutic compounds that can be used against influenza virus, atherapeutic compound of the present invention that has an anchoringdomain that binds heparin/heparan sulfate, the compound can be used inmammals (including humans) as well as avians.

Therapeutic Domain

A compound of the present invention includes at least one therapeuticdomain that has an extracellular activity that can prevent or impede theinfection of a cell by a pathogen, can modulate the immune response of asubject, or can improve transduction efficiency of a recombinant virus.The therapeutic activity can be, as nonlimiting examples, a bindingactivity, a catalytic activity, or an inhibitory activity. In someembodiments of the present invention, the therapeutic activity acts tomodify or inhibit a function of the pathogen that contributes toinfectivity of the cell by the pathogen. In other embodiments, atherapeutic domain can modify or inhibit a function of the target cellor target organism.

For example, the therapeutic domain can bind a receptor on a target cellthat is necessary for binding of the pathogen to a target cell. In thisway the therapeutic moiety can block binding of the pathogen to a targetcell and prevent infection. In an alternative, a therapeutic domain canbind a molecule or epitope on a pathogen to prevent an interaction ofthe molecule or epitope with a target cell that is necessary forinfection. A therapeutic domain can also have a catalytic activity thatcan degrade a molecule or epitope of the pathogen or host that allowsfor or promotes infection of a target cell by a host. In yet otherembodiments, a therapeutic domain can be an inhibitor of an activitythat is necessary for target cell infection by a pathogen. The inhibitedactivity can be an activity of the host organism or of the pathogen.

The therapeutic domain preferably acts extracellularly, meaning that itsinfection-preventing, inflammatory response-modulating, ortransduction-enhancing activity takes place at the target cell surfaceor in the immediate area surrounding the target cell, including siteswithin the extracellular matrix, intracellular spaces, or luminal spacesof tissues.

A therapeutic domain is preferably a peptide or protein domain(including a modified or derivatized peptide or protein domain), orcomprises a moiety coupled to a peptide or protein. A moiety coupled toa peptide or protein can be any type of molecule that can prevent orimpede the infection of a target cell by a pathogen, and is preferablyan organic molecule, such as, for example, nucleic acid, peptide nucleicacid, nucleic acid analogue, nucleotide, nucleotide analogue, smallorganic molecule, polymer, lipids, steroid, fatty acid, carbohydrate, orany combination of any of these.

A therapeutic domain can be a synthetic peptide or polypeptide, or cancomprise a synthetic molecule that can be conjugated to a peptide orpolypeptide, can be a naturally-occurring peptide or protein, or adomain of naturally-occurring protein. A therapeutic domain can also bea peptide or protein that is substantially homologous to anaturally-occurring peptide or protein.

A therapeutic domain can have utility in a particular species, or canprevent or impede pathogen infection in more than one species. Forexample, therapeutic domains that inhibit pathogen functions can ingeneral be used in a range of species that can be infected by the host,while therapeutic domains that interrupt host-pathogen interactions byinterfering with a property of the host may or may not bespecies-specific. In many cases, anchoring domains and therapeuticdomains can be effective in more than one species, so that compounds ofthe present invention can be used to advance human and animal health,while reducing propagation and spread of the virus through animal hosts.For example, when the therapeutic domain is a sialidase, a sialidasethat can cleave more than one type of linkage between a sialic acidresidue and the remainder of a substrate molecule, in particular, asialidase that can cleave both alpha(2,6)-Gal and alpha(2,3)-Gallinkages, can protect humans from infections by a broad-spectrum ofinfluenza viruses, including viruses that are naturally hosted indifferent species such as birds, pigs or horses.

Linkers

A compound of the present invention can optionally include one or morelinkers that can join domains of the compound. Linkers can be used toprovide optimal spacing or folding of the domains of a compound. Thedomains of a compound joined by linkers can be therapeutic domains,anchoring domains, or any other domains or moieties of the compound thatprovide additional functions such as enhancing compound stability,facilitating purification, etc. A linker used to join domains ofcompounds of the present invention can be a chemical linker or an aminoacid or peptide linker. Where a compound comprises more than one linker,the linkers can be the same or different. Where a compound comprisesmore than one linker, the linkers can be of the same or differentlengths.

Many chemical linkers of various compositions, polarity, reactivity,length, flexibility, and cleavability are known in the art of organicchemistry. Preferred linkers of the present invention include amino acidor peptide linkers. Peptide linkers are well known in the art.Preferably linkers are between one and one hundred amino acids inlength, and more preferably between one and thirty amino acids inlength, although length is not a limitation in the linkers of thecompounds of the present invention. Preferably linkers comprise aminoacid sequences that do not interfere with the conformation and activityof peptides or proteins encoded by monomers of the present invention.Some preferred linkers of the present invention are those that includethe amino acid glycine. For example, linkers having the sequence:

(GGGGS (SEQ ID NO:10))n, where n is a whole number between 1 and 20, ormore preferably between 1 and 12, can be used to link domains oftherapeutic compounds of the present invention.

The present invention also comprises nucleic acid molecules that encodeprotein-based compounds of the present invention that comprise at leastone therapeutic domain and at least one anchoring domain. The nucleicacid molecules can have codons optimized for expression in particularcell types, such as, for example E. coli or human cells. The nucleicacid molecules or the present invention that encode protein-basedcompounds of the present invention that comprise at least onetherapeutic domain and at least one anchoring domain can also compriseother nucleic acid sequences, including but not limited to sequencesthat enhance gene expression. The nucleic acid molecules can be invectors, such as but not limited to expression vectors.

Composition Comprising at Least One Anchoring Domain and at Least OneProtease Inhibitor

In some aspects of the present invention, a therapeutic domain that hasan extracellular activity that can prevent the infection of a cell by apathogen is a protease inhibitor. The protease inhibitor can be any typeof chemical entity, such as, for example, a carbohydrate or polymer, butis preferably a protein or peptide that inhibits the activity of anenzyme. Preferably, the protease inhibitor inhibits the activity of anenzyme that at least partially processes at least one pathogen or hostcell protein, where the processing of the pathogen or host cell proteinis necessary for pathogen infectivity. The enzyme that can process aviral protein necessary for pathogen infectivity can be a pathogenenzyme, or an enzyme that originates from the host organism. Preferably,the processing enzyme acts at or near the target cell surface, so that acompound of the present invention that is anchored at or near thesurface of a target cell can effectively inhibit the activity of theenzyme.

Compounds of the present invention that comprise protease inhibitorydomains can be used to inhibit infection by any pathogen that requires aprotease in its life cycle, in which the protease is active at or nearthe surface of the host cell. These protein-based compositions can have,for example, one of the following structures:

(Anchoring Domain)n-linker-(Protease Inhibitor)n (n=1, 2, 3 or more)

or:

(Protease Inhibitor)n-linker-(Anchoring Domain)n (n=1, 2, 3 or more)

The protease inhibitor can be a monomeric form of a peptide orpolypeptide or can be multiple copies of the same polypeptide that areeither linked directly or with spacing sequence in between.Alternatively, different polypeptide-based protease inhibitors can belinked with each other, such as, for example, aprotinin linked withsoybean protease inhibitor as protease inhibiting functional domains.The polypeptides or peptides can be linked directly or via a spacercomposed of peptide linker sequence. The anchoring domain can be anypeptide or polypeptide that can bind at or near the surface of targetcells.

The protease inhibitor can be a naturally occurring protease inhibitor(or an active portion thereof) or can be an engineered proteaseinhibitor. A peptide protease inhibitor used in a compound of thepresent invention can have a sequence substantially homologous to anaturally occurring protease inhibitor, having one or more deletions,additions, or substitutions while retaining the activity, orsubstantially retaining the same activity, of the naturally occurringprotease inhibitor.

In one preferred embodiment of the present invention, a therapeuticcompound of the present invention is for the prevention and treatment ofinfluenza in humans, and the therapeutic domain is a protein or peptideprotease inhibitor that can inhibit a serine protease that can cleavethe influenza virus hemagglutinin precursor protein HA0 into HA1 andHA2.

A number of serine protease inhibitors have been shown to reduce HAcleavage and influenza virus activation in cultured cells, in chickenembryos and in lungs of infected mice. They include many of the commonlyused trypsin inhibitors, such as: aprotinin (Zhirnov O P, Ikizler M Rand Wright P F. (2002) J Virol 76:8682-8689), leupeptin (Zhirnov O P,Ikizler M R and Wright P F. (2002) J Virol 76:8682-8689; Tashiro M,Klenk H D and Rott R. (1987) J Gen Virol 68:2039-2043), soybean proteaseinhibitor (Barbey-Morel C L, Oeltmann T N, Edwards K M and Wright P F.(1987) J Infect Dis 155:667-672), e-aminocaproic acid (Zhirnov O P,Ovchartenko A V and Bukrinskaya A G. 1982. Arch Virol 73:263-272) andn-p-tosyl-L-lysine chloromethylketone (TLCK) (Barbey-Morel C L, OeltmannT N, Edwards K M and Wright P F. (1987) J Infect Dis 155:667-672). Amongthese, aerosol inhalation of aprotinin has shown definitive therapeuticeffects against influenza and parainfluenza bronchopneumonia in mice(Zhirnov O P, Ovcharenko A V and Bukrinskaya A G. (1984) J Gen Virol65:191-196; Zhirnov O P, Ovcharenko A V and Bukrinskaya A G. (1985) JGen Virol 66:1633-1638; Zhirnov O P. (1987) J Med Virol 21:161-167;Ovcharenko A V and Zhirnov O P. (1994) Antiviral Res 23:107-118) as wellas in human (Zhirnov O P. (1983) Problems Virol. 4:9-12 (in Russian)).

Aprotinin (SEQ ID NO: 1; FIG. 1) is a 58 amino acid polypeptideinhibitor (also called Trasylol or bovine pancreatic trypsin inhibitor(BPTI)). A compound of the present invention can have one or moreaprotinin domains; for example, a therapeutic composition of the presentinvention can have from one to six aprotinin polypeptides, morepreferably from one to three aprotinin polypeptides. A compound of thepresent invention can also have a therapeutic domain comprising apolypeptide or peptide having substantial homology to the amino acidsequence of aprotinin.

A compound for preventing or treating influenza that comprises aprotease inhibitor preferably comprises an anchoring domain that canbind at or near the surface of epithelial cells. In some preferredembodiments, the epithelium anchoring domain is a GAG-binding sequencefrom a human protein, such as, for example, the GAG-binding sequence ofhuman platelet factor 4 (PF4) (SEQ ID NO:2), human interleukin 8 (IL8)(SEQ ID NO:3), human antithrombin III (AT III) (SEQ ID NO:4), humanapoprotein E (ApoE) (SEQ ID NO:5), human angio-associated migratory cellprotein (AAMP) (SEQ ID NO:6), or human amphiregulin (SEQ ID NO:7) (FIG.2). A compound of the present invention can also have an anchoringdomain comprising a polypeptide or peptide having substantial homologyto the amino acid sequences of the GAG-binding domains listed in SEQ IDNO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, and SEQ IDNO:7.

Clinically, a drug comprising aprotinin and an epithelial anchoringdomain can be administered by aerosol inhalation to cover the entirerespiratory tract to prevent and treat bronchopneumonia caused byinfluenza viruses, or any other virus, such as parainfluenza virus, thatrequires serine proteases in its life cycle. Alternatively, anaprotinin/epithelial anchoring domain fusion protein can be administeredas nasal spray to treat uncomplicated early stage influenza cases orother infections by respiratory viruses. In addition, anaprotinin/epithelial anchoring domain fusion protein can be used as aprophylaxis for influenza or other viral infections before an infectionoccurs.

Composition Comprising at Least One Anchoring Domain and at Least OneCatalytic Activity

In some aspects of the present invention, a therapeutic domain that hasan extracellular activity that can prevent the infection of a cell by apathogen is a catalytic activity. The enzymatic activity can be acatalytic activity that removes, degrades or modifies a host molecule orcomplex or a pathogen molecule or complex that contributes to theinfectivity of the pathogen. Preferably the host molecule or complex orpathogen molecule or complex that is removed, degraded, or modified bythe enzymatic activity of a compound of the present invention is on, at,or near the surface of a target cell, so that a compound of the presentinvention that is anchored to the surface of a target cell caneffectively inhibit the host or pathogen molecule or complex.

For example, a therapeutic domain can have a catalytic activity that candigest a molecule or epitope of the pathogen or target cell that isrequired for host-pathogen binding, and subsequent entry of the pathogeninto the target cell. Receptors on target cells that allow for the entryof viruses into cells can be the target of an enzymatic activity of acompound of the present invention.

Compounds of the present invention that comprise catalytic domains canbe used to inhibit infection by any pathogen that uses a receptor togain entry to a target cell, as long as removal of the receptor does notimpair the organism. These protein-based compositions can have, forexample, one of the following structures:

(Anchoring Domain)n-[linker]-(Enzymatic Activity)n (n=1, 2, 3 or more)

-   -   or:

(Enzymatic Activity)n (n=1, 2, 3 or more)-[linker]-(Anchoring Domain)n,

-   -   where the linkers are optional.

The enzymatic activity can be a monomeric form of a peptide orpolypeptide or can be multiple copies of the same polypeptide that areeither linked directly or with spacing sequence in between. Thepolypeptides or peptides can be linked directly or via a spacer composedof peptide linker sequence. The anchoring domain can be any peptide orpolypeptide that can bind to or near the surface of target cells.

In one preferred embodiment of the present invention, a therapeuticdomain comprises a sialidase that can eliminate or greatly reduce thelevel of sialic acid on the surface of epithelial cells. Sialic acid isa receptor for influenza viruses. Thus, treating the surface ofrespiratory epithelial cells with a sialidase can prevent influenzainfections or interrupt early infections. The therapeutic domain cancomprise a complete sialidase protein, or an active portion thereof.Sialic acid is a receptor for influenza viruses, and at least one of thereceptors for parainfluenza virus, some coronavirus and rotavirus,Streptococcus pneumoniae, Mycoplasma pneumoniae, Haemophilus influenzae,Moraxella catarrhalis, Pseudomonas aeruginosa, and Helicobacter pylori.Thus, treating the surface of respiratory epithelial cells with asialidase can prevent influenza or other viral infections or interruptearly infections, as well as prevent or reduce colonization of bacteriasuch as Streptococcus pneumoniae, Mycoplasma pneumoniae, Haemophilusinfluenzae, Moraxella catarrhalis, and Pseudomonas aeruginosa. Treatingthe gastrointestinal epithelial cells with a sialidase can prevent orreduce colonization of Helicobacter pylori in the stomach.

Sialic acid also mediates cell adhesion and interactions betweeninflammatory cells and target cells. Therefore, treating the surface ofrespiratory epithelial cells with a sialidase can prevent therecruitment of inflammatory cells to the airway surface, and thereforecan treat allergic reactions including asthma and allergic rhinitis.

Since sialic acid serves as a barrier that hinder cell entry by a genetherapy vector, treating the target cells with a sialidase can increasetransduction efficiency, and therefore improve efficacy of the genetherapy.

Preferred sialidases are the large bacterial sialidases that can degradethe receptor sialic acids Neu5Ac alpha(2,6)-Gal and Neu5Acalpha(2,3)-Gal. For example, the bacterial sialidase enzymes fromClostridium perfringens (Genbank Accession Number X87369), Actinomycesviscosus, Arthrobacter ureafaciens, or Micromonospora viridifaciens(Genbank Accession Number D01045) can be used. Therapeutic domains ofcompounds of the present invention can comprise all or a portion of theamino acid sequence of a large bacterial sialidase or can comprise aminoacid sequences that are substantially homologous to all or a portion ofthe amino acid sequence of a large bacterial sialidase. In one preferredembodiment, a therapeutic domain comprises a sialidase encoded byActinomyces viscosus, such as that of SEQ ID NO:12, or such as sialidasesequence substantially homologous to SEQ ID NO:12. In yet anotherpreferred embodiment, a therapeutic domain comprises the catalyticdomain of the Actinomyces viscosus sialidase extending from amino acids274-666 of SEQ ID NO:12, or a substantially homologous sequence.

Other preferred sialidases are the human sialidases such as thoseencoded by the genes NEU2 (SEQ ID NO:8; Genbank Accession Number Y16535;Monti, E, Preti, Rossi, E., Ballabio, A and Borsani G. (1999) Genomics57:137-143) and NEU4 (SEQ ID NO:9; Genbank Accession Number NM080741;Monti, E, Preti, A, Venerando, B and Borsani, G. (2002) Neurochem Res27:646-663) (FIG. 3). Therapeutic domains of compounds of the presentinvention can comprise all or a portion of the amino acid sequences of ahuman sialidase or can comprise amino acid sequences that aresubstantially homologous to all or a portion of the amino acid sequencesof a human sialidase. Preferably, where a therapeutic domain comprises aportion of the amino acid sequences of a naturally occurring sialidase,or sequences substantially homologous to a portion of the amino acidsequences of a naturally occurring sialidase, the portion comprisesessentially the same activity as the human sialidase.

A compound for preventing or treating influenza that comprises anenzymatic domain preferably comprises an anchoring domain that can bindat or near the surface of epithelial cells. In some preferredembodiments, the epithelium-anchoring domain is a GAG-binding sequencefrom a human protein, such as, for example, the GAG-binding amino acidsequences of human platelet factor 4 (PF4) (SEQ ID NO:2), humaninterleukin 8 (IL8) (SEQ ID NO:3), human antithrombin III (AT III) (SEQID NO:4), human apoprotein E (ApoE) (SEQ ID NO:5), humanangio-associated migratory cell protein (AAMP) (SEQ ID NO:6), and humanamphiregulin (SEQ ID NO:7) (FIG. 2). An epithelial anchoring domain canalso be substantially homologous to a naturally occurring GAG-bindingsequence, such as those listed in FIG. 2.

It is also within the scope of the present invention to use compoundscomprising a human sialidase, or comprising a sialidase with substantialhomology to a sialidase, in the absence of an anchoring domain, in thetreatment or prevention of pathogen infections, such as but not limitedto influenza, paramyxovirus, coronavirus, rotavirus, and Pseudomonasaeruginosa infections or bacterial infections; in the treatment orprevention of allergic and inflammatory responses, and to improve thetransduction efficiency of a recombinant virus.

The present invention recognizes that such infections may be preventedor abated by the use of sialidases, such as, but not limited to, the A.viscosus sialidase or human sialidases such as NEU2 and NEU4. Thesialidases can optionally be adapted, by genetic or chemicalengineering, or by pharmaceutical formulation, to improve their halflife or retention at the respiratory epithelium.

Because influenza viruses primarily infect the upper respiratory tract,removing the receptor sialic acid locally in the nasal cavity andnasopharynx area can prevent infections or interrupt early infections.The sialidase can be delivered to the upper respiratory tract as a nasalspray, and it can be used either in therapeutic mode during early stageof influenza (or other infection) or in prophylactic mode before theinfection occurs. Alternatively, it can be delivered to the lowerrespiratory tract as an inhalant to treat influenza and to preventinfluenza complications, such as bronchopneumonia.

II. Therapeutic Composition Comprising at Least One Sialidase Activity

The present invention includes a therapeutic composition that comprisesat least one sialidase activity. The sialidase activity can be asialidase isolated from any source, such as, for example, a bacterial ormammalian source, or can be a recombinant protein that is substantiallyhomologous to at least a portion of a naturally occurring sialidase.Preferred sialidases are the large bacterial sialidases that can degradethe receptor sialic acids Neu5Ac alpha(2,6)-Gal and Neu5Acalpha(2,3)-Gal. For example, the bacterial sialidase enzymes fromClostridium perfringens (Genbank Accession Number X87369), Actinomycesviscosus (Genbank Accession Number L06898), Arthrobacter ureafaciens, orMicromonospora viridifaciens (Genbank Accession Number D01045) orsubstantially homologous proteins can be used.

For example, therapeutic compounds of the present invention can comprisea large bacterial sialidase or can comprise a protein with the aminoacid sequence of a large bacterial sialidase or can comprise amino acidsequences that are substantially homologous to the amino acid sequenceof a large bacterial sialidase. A preferred pharmaceutical compositionof the present invention comprises the A. viscosus sialidase (SEQ IDNO:12), or comprises a protein substantially homologous to the A.viscosus sialidase.

Other preferred sialidases are the human sialidases such as thoseencoded by the genes NEU2 (SEQ ID NO:8; Genbank Accession Number Y16535;Monti, E, Preti, Rossi, E., Ballabio, A and Borsani G. (1999) Genomics57:137-143) and NEU4 (SEQ ID NO:9; Genbank Accession Number NM080741;Monti, E, Preti, A, Venerando, B and Borsani, G. (2002) Neurochem Res27:646-663) (FIG. 3). Therapeutic domains of compounds of the presentinvention can comprise a human sialidase protein that is substantiallyhomologous to the amino acid sequences of a human sialidase or cancomprise amino acid sequences that are substantially homologous to allor a portion of the amino acid sequences of a human sialidase.Preferably, where a therapeutic domain comprises a portion of the aminoacid sequences of a naturally occurring sialidase, or sequencessubstantially homologous to a portion of the amino acid sequences of anaturally occurring sialidase, the portion comprises essentially thesame activity as the human sialidase.

A pharmaceutical composition comprising a sialidase can include othercompounds, including but not limited to other proteins, that can alsohave therapeutic activity. A pharmaceutical composition comprising asialidase can include other compounds that can enhance the stability,solubility, packaging, delivery, consistency, taste, or fragrance of thecomposition.

A pharmaceutical composition comprising a sialidase can be formulatedfor nasal, tracheal, bronchial, oral, or topical administration, or canbe formulated as an injectable solution or as eyedrops. A pharmaceuticalcomposition comprising a sialidase can be used to treat or preventpathogen infection, to treat or prevent allergy or inflammatoryresponse, or to enhance the transduction efficiency of a recombinantvirus for gene therapy.

III. Sialidase Catalytic Domain Proteins

The present invention also includes sialidase catalytic domain proteins.As used herein a “sialidase catalytic domain protein” comprises acatalytic domain of a sialidase but does not comprise the entire aminoacid sequence of the sialidase from which the catalytic domain isderived. A sialidase catalytic domain protein has sialidase activity.Preferably, a sialidase catalytic domain protein comprises at least 10%,at least 20%, at least 50%, at least 70% of the activity of thesialidase from which the catalytic domain sequence is derived. Morepreferably, a sialidase catalytic domain protein comprises at least 90%of the activity of the sialidase from which the catalytic domainsequence is derived.

A sialidase catalytic domain protein can include other amino acidsequences, such as but not limited to additional sialidase sequences,sequences derived from other proteins, or sequences that are not derivedfrom sequences of naturally-occurring proteins. Additional amino acidsequences can perform any of a number of functions, includingcontributing other activities to the catalytic domain protein, enhancingthe expression, processing, folding, or stability of the sialidasecatalytic domain protein, or even providing a desirable size or spacingof the protein.

A preferred sialidase catalytic domain protein is a protein thatcomprises the catalytic domain of the A. viscosus sialidase. Preferably,an A. viscosus sialidase catalytic domain protein comprises amino acids270-666 of the A. viscosus sialidase sequence (SEQ ID NO:12).Preferably, an A. viscosus sialidase catalytic domain protein comprisesan amino acid sequence that begins at any of the amino acids from aminoacid 270 to amino acid 290 of the A. viscosus sialidase sequence (SEQ IDNO:12) and ends at any of the amino acids from amino acid 665 to aminoacid 901 of said A. viscosus sialidase sequence (SEQ ID NO:12), andlacks any A. viscosus sialidase protein sequence extending from aminoacid 1 to amino acid 269. (As used herein “lacks any A. viscosussialidase protein sequence extending from amino acid 1 to amino acid269” means lacks any stretch of four or more consecutive amino acids asthey appear in the designated protein or amino acid sequence.)

In some preferred embodiments, an A. viscosus sialidase catalytic domainprotein comprises amino acids 274-681 of the A. viscosus sialidasesequence (SEQ ID NO:12) and lacks other A. viscosus sialidase sequence.In some preferred embodiments, an A. viscosus sialidase catalytic domainprotein comprises amino acids 274-666 of the A. viscosus sialidasesequence (SEQ ID NO:12) and lacks any other A. viscosus sialidasesequence. In some preferred embodiments, an A. viscosus sialidasecatalytic domain protein comprises amino acids 290-666 of the A.viscosus sialidase sequence (SEQ ID NO:12) and lacks any other A.viscosus sialidase sequence. In yet other preferred embodiments, an A.viscosus sialidase catalytic domain protein comprises amino acids290-681 of the A. viscosus sialidase sequence (SEQ ID NO:12) and lacksany other A. viscosus sialidase sequence.

The present invention also comprises nucleic acid molecules that encodeprotein-based compounds of the present invention that comprise acatalytic domain of a sialidase. The nucleic acid molecules can havecodons optimized for expression in particular cell types, such as, forexample E. coli or human cells. The nucleic acid molecules or thepresent invention that encode protein-based compounds of the presentinvention that comprise at least one catalytic domain of a sialidase canalso comprise other nucleic acid sequences, including but not limited tosequences that enhance gene expression. The nucleic acid molecules canbe in vectors, such as but not limited to expression vectors.

Fusion Proteins

Sialidase catalytic domain proteins can be fusion proteins, in which thefusion protein comprises at least one sialidase catalytic domain and atleast one other protein domain, including but not limited to: apurification domain, a protein tag, a protein stability domain, asolubility domain, a protein size-increasing domain, a protein foldingdomain, a protein localization domain, an anchoring domain, anN-terminal domain, a C-terminal domain, a catalytic activity domain, abinding domain, or a catalytic activity-enhancing domain. Preferably,the at least one other protein domain is derived from another source,such as, but not limited to, sequences from another protein. The atleast one other protein domain need not be based on any known proteinsequence, but can be engineered and empirically tested to perform anyfunction in the fusion protein.

Purification domains can include, as nonlimiting examples, one or moreof a his tag, a calmodulin binding domain, a maltose binding proteindomain, a streptavidin domain, a streptavidin binding domain, an inteindomain, or a chitin binding domain. Protein tags can comprise sequencesthat can be used for antibody detection of proteins, such as, forexample, the myc tag, the hemagglutinin tag, or the FLAG tag. Proteindomains that enhance protein expression, modification, folding,stability, size, or localization can be based on sequences of knowproteins or engineered. Other protein domains can have binding orcatalytic activity or enhance the catalytic activity of the sialidasecatalytic domain.

Preferred fusion proteins of the present invention comprise at least onesialidase catalytic domain and at least one anchoring domain. Preferredanchoring domains include GAG-binding domains, such as the GAG-bindingdomain or human amphiregulin (SEQ ID NO:7).

Sialidase catalytic domains and other domains of a fusion protein of thepresent invention can optionally be joined by linkers, such as but notlimited to peptide linkers. A variety of peptide linkers are known inthe art. A preferred linker is a peptide linker comprising glycine, suchas G-G-G-G-S (SEQ ID NO:10).

The present invention also comprises nucleic acid molecules that fusionproteins of the present invention that comprise a catalytic domain of asialidase. The nucleic acid molecules can have codons optimized forexpression in particular cell types, such as, for example E. coli orhuman cells. The nucleic acid molecules or the present invention thatencode fusion proteins of the present invention can also comprise othernucleic acid sequences, including but not limited to sequences thatenhance gene expression. The nucleic acid molecules can be in vectors,such as but not limited to expression vectors.

IV Pharmaceutical Compositions

The present invention includes compounds of the present inventionformulated as pharmaceutical compositions. The pharmaceuticalcompositions comprise a pharmaceutically acceptable carrier prepared forstorage and preferably subsequent administration, which have apharmaceutically effective amount of the compound in a pharmaceuticallyacceptable carrier or diluent. Acceptable carriers or diluents fortherapeutic use are well known in the pharmaceutical art, and aredescribed, for example, in Remington's Pharmaceutical Sciences, 18thEd., Mack Publishing Co., Easton, Pa. (1990)). Preservatives,stabilizers, dyes and even flavoring agents can be provided in thepharmaceutical composition. For example, sodium benzoate, sorbic acidand esters of p-hydroxybenzoic acid can be added as preservatives. Inaddition, antioxidants and suspending agents can be used.

Depending on the target cell, the compounds of the present invention canbe formulated and used as tablets, capsules or elixirs for oraladministration; salves or ointments for topical application;suppositories for rectal administration; sterile solutions, suspensions,and the like for use as inhalants or nasal sprays. Injectables can alsobe prepared in conventional forms either as liquid solutions orsuspensions, solid forms suitable for solution or suspension in liquidprior to injection, or as emulsions. Suitable excipients are, forexample, water, saline, dextrose, mannitol, lactose, lecithin, albumin,sodium glutamate, cysteine hydrochloride and the like. In addition, ifdesired, the injectable pharmaceutical compositions can contain minoramounts of nontoxic auxiliary substances, such as wetting agents, pHbuffering agents and the like.

The pharmaceutically effective amount of a test compound required as adose will depend on the route of administration, the type of animal orpatient being treated, and the physical characteristics of the specificanimal under consideration. The dose can be tailored to achieve adesired effect, but will depend on such factors as weight, diet,concurrent medication and other factors which those skilled in themedical arts will recognize. In practicing the methods of the presentinvention, the pharmaceutical compositions can be used alone or incombination with one another, or in combination with other therapeuticor diagnostic agents. These products can be utilized in vivo, preferablyin a mammalian patient, preferably in a human, or in vitro. In employingthem in vivo, the pharmaceutical compositions can be administered to thepatient in a variety of ways, including topically, parenterally,intravenously, subcutaneously, intramuscularly, colonically, rectally,nasally or intraperitoneally, employing a variety of dosage forms. Suchmethods can also be used in testing the activity of test compounds invivo.

In preferred embodiments, these pharmaceutical compositions may be inthe form of orally-administrable suspensions, solutions, tablets orlozenges; nasal sprays; inhalants; injectables, topical sprays,ointments, powders, or gels.

When administered orally as a suspension, compositions of the presentinvention are prepared according to techniques well-known in the art ofpharmaceutical formulation and may contain microcrystalline cellulosefor imparting bulk, alginic acid or sodium alginate as a suspendingagent, methylcellulose as a viscosity enhancer, and sweeteners/flavoringagents known in the art. As immediate release tablets, thesecompositions may contain microcrystalline cellulose, dicalciumphosphate, starch, magnesium stearate and lactose and/or otherexcipients, binders, extenders, disintegrants, diluents and lubricantsknown in the art. Components in the formulation of a mouthwash or rinseinclude antimicrobials, surfactants, cosurfactants, oils, water andother additives such as sweeteners/flavoring agents known in the art.

When administered by a drinking solution, the composition comprises oneor more of the compounds of the present invention, dissolved in water,with appropriate pH adjustment, and with carrier. The compound may bedissolved in distilled water, tap water, spring water, and the like. ThepH can preferably be adjusted to between about 3.5 and about 8.5.Sweeteners may be added, e.g., 1% (w/v) sucrose.

Lozenges can be prepared according to U.S. Pat. No. 3,439,089, hereinincorporated by reference for these purposes.

When administered by nasal aerosol or inhalation, the pharmaceuticalcompositions are prepared according to techniques well-known in the artof pharmaceutical formulation and may be prepared as solutions insaline, employing benzyl alcohol or other suitable preservatives,absorption promoters to enhance bioavailability, fluorocarbons, and/orother solubilizing or dispersing agents known in the art. See, forexample, Ansel, H. C. et al., Pharmaceutical Dosage Forms and DrugDelivery Systems, Sixth Ed. (1995). Preferably these compositions andformulations are prepared with suitable nontoxic pharmaceuticallyacceptable ingredients. These ingredients are known to those skilled inthe preparation of nasal dosage forms and some of these can be found inRemington's Pharmaceutical Sciences, 18th Ed., Mack Publishing Co.,Easton, Pa. (1990, a standard reference in the field. The choice ofsuitable carriers is highly dependent upon the exact nature of the nasaldosage form desired, e.g., solutions, suspensions, ointments, or gels.Nasal dosage forms generally contain large amounts of water in additionto the active ingredient. Minor amounts of other ingredients such as pHadjusters, emulsifiers or dispersing agents, preservatives, surfactants,jelling agents, or buffering and other stabilizing and solubilizingagents may also be present. Preferably, the nasal dosage form should beisotonic with nasal secretions.

Nasal formulations can be administers as drops, sprays, aerosols or byany other intranasal dosage form. Optionally, the delivery system can bea unit dose delivery system. The volume of solution or suspensiondelivered per dose can preferably be anywhere from about 5 to about 2000microliters, more preferably from about 10 to about 1000 microliters,and yet more preferably from about 50 to about 500 microliters. Deliverysystems for these various dosage forms can be dropper bottles, plasticsqueeze units, atomizers, nebulizers or pharmaceutical aerosols ineither unit dose or multiple dose packages.

The formulations of this invention may be varied to include; (1) otheracids and bases to adjust the pH; (2) other tonicity imparting agentssuch as sorbitol, glycerin and dextrose; (3) other antimicrobialpreservatives such as other parahydroxy benzoic acid esters, sorbate,benzoate, propionate, chlorbutanol, phenylethyl alcohol, benzalkoniumchloride, and mercurials; (4) other viscosity imparting agents such assodium carboxymethylcellulose, microcrystalline cellulose,polyvinylpyrrolidone, polyvinyl alcohol and other gums; (5) suitableabsorption enhancers; (6) stabilizing agents such as antioxidants, likebisulfite and ascorbate, metal chelating agents such as sodium edetateand drug solubility enhancers such as polyethylene glycols.

V. Method of Preventing or Treating Infection by a Pathogen

The present invention also includes methods of preventing or treatinginfection by a pathogen. In one aspect, the method includes: treating asubject that is infected with a pathogen or at risk of being infectedwith a pathogen with a pharmaceutical composition of the presentinvention that comprises a compound that comprises at least oneanchoring domain that can anchor the compound at or near the surface ofa target cell and at least one therapeutic domain comprising a peptideor protein that has at least one extracellular activity that can preventthe infection of a target cell by a pathogen. In some preferredembodiments, the method includes applying a therapeutically effectiveamount of a pharmaceutical composition of the present invention toepithelial cells of a subject. The subject to be treated can be ananimal or human subject.

In another aspect, the method includes: treating a subject that isinfected with a pathogen or at risk of being infected with a pathogenwith a pharmaceutical composition of the present invention thatcomprises a protein-based compound that comprises a sialidase activity.In some preferred embodiments, the method includes applying atherapeutically effective amount of a pharmaceutical composition of thepresent invention to epithelial cells of a subject. The sialidaseactivity can be an isolated naturally occurring sialidase protein, or arecombinant protein substantially homologous to at least a portion of anaturally occurring sialidase. A preferred pharmaceutical compositioncomprises a sialidase with substantial homology to the A. viscosussialidase (SEQ ID NO:12). The subject to be treated can be an animal orhuman subject.

In yet another aspect, the method includes: treating a subject that isinfected with a pathogen or at risk of being infected with a pathogenwith a pharmaceutical composition of the present invention thatcomprises a protein-based compound that comprises a sialidase catalyticdomain. In some preferred embodiments, the method includes applying atherapeutically effective amount of a pharmaceutical composition of thepresent invention to epithelial cells of a subject. The sialidasecatalytic domain is preferably can substantially homologous to thecatalytic domain of a naturally occurring sialidase. A preferredpharmaceutical composition comprises a sialidase catalytic domain withsubstantial homology to amino acids 274-666 the A. viscosus sialidase(SEQ ID NO:12). The subject to be treated can be an animal or humansubject.

A pathogen can be a viral, bacterial, or protozoan pathogen. In someembodiments, the pathogen is one of the following: influenza viruses,parainfluenza virus, respiratory syncytial virus (RSV), coronavirus,rotavirus, Streptococcus pneumoniae, Mycoplasma pneumoniae, Haemophilusinfluenzae, Moraxella catarrhalis, Pseudomonas aeruginosa, andHelicobacter pylori. In one preferred embodiment, the pathogen isinfluenza virus.

Compounds of the present invention can be designed for human use oranimal use. In some aspects of the present invention, a compound of thepresent invention can be used to prevent pathogen infection in a classof animals, such as mammals. In some aspects of the present invention, acomposition can be used for human and animal use (although theformulation may differ). In these aspects, the active domains of acompound can be effective against more than one pathogen species, type,subtype, or strain and can be active in more than one host species. Forexample, some preferred compounds of the present invention thatcomprise, for example, active domains such as protease inhibitors thatprevent processing of the HA protein of influenza virus, or sialidasesthat remove sialic acid receptors from target cells, or anchoringdomains such as domains that bind heparin or heparan sulfate, can beused in birds, mammals, or humans. Such compounds that can be effectiveagainst a range of pathogens with the capacity to infect different hostspecies can also be used in humans to combat infection by pathogens thatare naturally hosted in other species.

In some preferred embodiments of the present invention, thepharmaceutical composition prevents infection by influenza, and atherapeutically effective amount of the pharmaceutical composition isapplied to the respiratory epithelial cells of a subject. This can bedone by the use of an inhaler, or by the use of a nasal spray.Preferably, the inhaler or nasal spray is used from one to four times aday.

Because influenza viruses primarily infect the upper respiratory tract,removing the receptor sialic acid locally in the nasal cavity, pharynx,trachea and bronchi can prevent infections or interrupt earlyinfections. The sialidase can be delivered to the upper respiratorytract as a nasal spray or as an inhalant, and it can be used either intherapeutic mode during early stage of influenza (or other infection) orin prophylactic mode before the infection occurs. Alternatively, it canbe delivered to the lower respiratory tract as an inhalant to treatinfluenza and to prevent influenza complications, such asbronchopneumonia. Similarly, the sialidase can be delivered as nasalspray or inhalant to prevent or reduce infection by parainfluenza virusand coronavirus. It can also be delivered as an inhalant or nasal sprayto prevent or reduce airway colonization by pathogenic bacteria,including Streptococcus pneumoniae, Mycoplasma pneumoniae, Haemophilusinfluenzae, Moraxella catarrhalis and Pseudomonas aeruginosa. Thetherapeutic compounds can optionally be adapted, by genetic or chemicalengineering, or by pharmaceutical formulation, to improve theirhalf-life or retention at the respiratory epithelium. Additionally, itcan be delivered topically to the eyes or to surgical wounds in the formof drops, sprays or ointments to prevent and treat bacterial infectionincluding infection by Pseudomonas aeruginosa. It can also beadministered orally to treat infection by Helicobacter pylori.

Dosage

As will be readily apparent to one skilled in the art, the useful invivo dosage to be administered and the particular mode of administrationwill vary depending upon the age, weight and type of patient beingtreated, the particular pharmaceutical composition employed, and thespecific use for which the pharmaceutical composition is employed. Thedetermination of effective dosage levels, that is the dose levelsnecessary to achieve the desired result, can be accomplished by oneskilled in the art using routine methods as discussed above. Innon-human animal studies, applications of the pharmaceuticalcompositions are commenced at higher dose levels, with the dosage beingdecreased until the desired effect is no longer achieved or adverse sideeffects are reduced or disappear. The dosage for a compound of thepresent invention can range broadly depending upon the desired affects,the therapeutic indication, route of administration and purity andactivity of the compound. Typically, human clinical applications ofproducts are commenced at lower dosage levels, with dosage level beingincreased until the desired effect is achieved. Alternatively,acceptable in vitro studies can be used to establish useful doses androutes of administration of the test compound. Typically, dosages can bebetween about 1 ng/kg and about 10 mg/kg, preferably between about 10ng/kg and about 1 mg/kg, and more preferably between about 100 ng/kg andabout 100 micrograms/kg.

The exact formulation, route of administration and dosage can be chosenby the individual physician in view of the patient's condition (see,Fingle et al., in The Pharmacological Basis of Therapeutics (1975)). Itshould be noted that the attending physician would know how to and whento terminate, interrupt or adjust administration due to toxicity, organdysfunction or other adverse effects. Conversely, the attendingphysician would also know to adjust treatment to higher levels if theclinical response were not adequate. The magnitude of an administrateddoes in the management of the disorder of interest will vary with theseverity of the condition to be treated and to the route ofadministration. The severity of the condition may, for example, beevaluated, in part, by standard prognostic evaluation methods. Further,the dose and perhaps dose frequency, will also vary according to theage, body weight and response of the individual patient, including thosefor veterinary applications.

Thus, in accordance with the present invention, there is furtherprovided a method of treating and a pharmaceutical composition fortreating influenza virus infection and prevention of influenza virusinfection. The treatment involves administering to a patient in need ofsuch treatment a pharmaceutical carrier and a therapeutically effectiveamount of any composition of the present invention, or apharmaceutically acceptable salt thereof.

In one preferred regimen, appropriate dosages are administered to eachpatient by either inhaler, nasal spray, or by oral lozenge. It will beunderstood, however, that the specific dose level and frequency ofdosage for any particular patient may be varied and will depend upon avariety of factors including the activity of the specific salt or otherform employed, the metabolic stability and length of action of thatcompound, the age, body weight, general health, sex, diet, mode and timeof administration, rate of excretion, drug combination, the severity ofthe particular condition, and the host undergoing therapy.

VI. Method of Reducing, Preventing, or Treating Allergic andInflammatory Responses

The present invention also includes methods of reducing, preventing, ortreating an allergic or inflammatory response of a subject.

In one aspect, the method includes: preventing or treating an allergicor inflammatory response of a subject with a pharmaceutical compositionof the present invention that comprises a protein-based compound thatcomprises a sialidase activity. In some preferred embodiments, themethod includes applying a therapeutically effective amount of apharmaceutical composition of the present invention to epithelial cellsof a subject. The sialidase activity can be an isolated naturallyoccurring sialidase protein, or a recombinant protein substantiallyhomologous to at least a portion of a naturally occurring sialidase. Apreferred pharmaceutical composition comprises a sialidase withsubstantial homology to the A. viscosus sialidase (SEQ ID NO:12). Thesubject to be treated can be an animal or human subject.

In yet another aspect, the method includes: preventing or treating anallergic or inflammatory response of a subject with a pharmaceuticalcomposition of the present invention that comprises a protein-basedcompound that comprises a sialidase catalytic domain. In some preferredembodiments, the method includes applying a therapeutically effectiveamount of a pharmaceutical composition of the present invention toepithelial cells of a subject. The sialidase catalytic domain ispreferably can substantially homologous to the catalytic domain of anaturally occurring sialidase. A preferred pharmaceutical compositioncomprises a sialidase catalytic domain with substantial homology toamino acids 274-666 the A. viscosus sialidase (SEQ ID NO:12). Thesubject to be treated can be an animal or human subject.

The allergic or inflammatory response can be and acute or chroniccondition, and can include, as nonlimiting examples, asthma, otherallergic responses causing respiratory distress, allergic rhinitis,eczema, psoriasis, reactions to plant or animal toxins, or autoimmuneconditions.

In some preferred embodiments, compounds of the present invention can bedelivered as an inhalant or nasal spray to prevent or treat inflammationin the airway including, but not limited to, asthma and allergicrhinitis. Compounds of the present invention comprising sialidaseactivity (including sialidase catalytic domain proteins and sialidasefusion proteins) can also be administered as eye drops, ear drops, orsprays, ointments, lotions, or gels to be applied to the skin. Inanother aspect, the method includes treating a patient who hasinflammatory diseases with the present invention that comprises asialidase activity that is administered intravenously or as a localinjection.

Dosage

As will be readily apparent to one skilled in the art, the useful invivo dosage to be administered and the particular mode of administrationwill vary depending upon the age, weight and type of patient beingtreated, the particular pharmaceutical composition employed, and thespecific use for which the pharmaceutical composition is employed. Thedetermination of effective dosage levels, that is the dose levelsnecessary to achieve the desired result, can be accomplished by oneskilled in the art using routine methods as discussed above. Innon-human animal studies, applications of the pharmaceuticalcompositions are commenced at higher dose levels, with the dosage beingdecreased until the desired effect is no longer achieved or adverse sideeffects are reduced or disappear. The dosage for a compound of thepresent invention can range broadly depending upon the desired affects,the therapeutic indication, route of administration and purity andactivity of the compound. Typically, human clinical applications ofproducts are commenced at lower dosage levels, with dosage level beingincreased until the desired effect is achieved. Alternatively,acceptable in vitro studies can be used to establish useful doses androutes of administration of the test compound. Typically, dosages can bebetween about 1 ng/kg and about 10 mg/kg, preferably between about 10ng/kg and about 1 mg/kg, and more preferably between about 100 ng/kg andabout 100 micrograms/kg.

The exact formulation, route of administration and dosage can be chosenby the individual physician in view of the patient's condition (see,Fingle et al., in The Pharmacological Basis of Therapeutics (1975)). Itshould be noted that the attending physician would know how to and whento terminate, interrupt or adjust administration due to toxicity, organdysfunction or other adverse effects. Conversely, the attendingphysician would also know to adjust treatment to higher levels if theclinical response were not adequate. The magnitude of an administrateddoes in the management of the disorder of interest will vary with theseverity of the condition to be treated and to the route ofadministration. The severity of the condition may, for example, beevaluated, in part, by standard prognostic evaluation methods. Further,the dose and perhaps dose frequency, will also vary according to theage, body weight and response of the individual patient, including thosefor veterinary applications.

In some preferred regimens, appropriate dosages are administered to eachpatient by either inhaler, nasal spray, or by topical application. Itwill be understood, however, that the specific dose level and frequencyof dosage for any particular patient may be varied and will depend upona variety of factors including the activity of the specific salt orother form employed, the metabolic stability and length of action ofthat compound, the age, body weight, general health, sex, diet, mode andtime of administration, rate of excretion, drug combination, theseverity of the particular condition, and the host undergoing therapy.

VII. Method of Enhancing Gene Delivery by a Recombinant Viral Vector

The present invention also includes methods of gene delivery by arecombinant viral vector. In one aspect, the method includes:administering an effective amount of a compound of the present inventionthat comprises a protein having sialidase activity to at least one cellprior to or concomitant with the administration of at least onerecombinant viral vector. A composition of the present invention can beprovided in the same formulation as at least one recombinant viralvector, or in a separate formulation.

In some preferred embodiments, the method includes applying atherapeutically effective amount of a composition of the presentinvention and a recombinant viral vector to cells of a subject. Thesubject to be treated can be an animal or human subject. In aparticularly preferred embodiment, a recombinant viral vector is used totransduce epithelial target cells of a subject for gene therapy. Forexample, a recombinant viral vector can be used to transduce airwayepithelial cells of a subject with cystic fibrosis. In this case, acompound of the present invention can be administered by use of aninhaler. A recombinant virus comprising a therapeutic gene can beadministered concurrently or separately.

In other embodiments, cells can be treated with a compound of thepresent invention and a recombinant viral vector in vitro or “ex vivo”(that is, cells removed from a subject to be transplanted into a subjectafter transduction).

The sialidase activity can be an isolated naturally occurring sialidaseprotein, or a recombinant protein substantially homologous to at least aportion of a naturally occurring sialidase, including a sialidasecatalytic domain. A preferred pharmaceutical composition comprises asialidase with substantial homology to the A. viscosus sialidase (SEQ IDNO:12).

A compound of the present invention can be administered to target cellsfrom one day before to two hours subsequent to the administration of therecombinant virus. Preferably a compound of the present invention isadministered to target cells from four hours to ten minutes beforeadministration of the recombinant virus. Administration can be

A recombinant virus is preferably a recombinant virus that can be usedto transfer genes to mammalian cells, such as, preferably human cells.For example, a recombinant virus can be a retrovirus (includinglentivirus), adenovirus, adeno-associated virus (AAV) or herpes simplexvirus type 1. The recombinant virus comprises at least one exogenousgene that is to be transferred to a target cell. The gene is preferablya therapeutic gene, but this need not be the case. For example, the genecan be a gene used to mark cells or confer drug resistance.

In a preferred embodiment, the present invention includes methods ofimproving efficacy of a gene therapy vector. The method includestreating a patient with a compound of the present invention thatcomprises a sialidase activity and, in the same or a separate formation,with a recombinant virus. The compound of the present invention havingsialidase activity can be administered to the patient prior to,concomitant to, or even subsequent to the administration of arecombinant virus. In one embodiment, the sialidase is substantiallyhomologous to the Actinomyces viscosus sialidase (SEQ ID NO:12) or aportion thereof. In one preferred embodiment, the sialidase comprisesthe catalytic domain of the Actinomyces viscosus sialidase. In anotherembodiment, the recombinant virus is AAV. In yet another embodiment, thedisease is cystic fibrosis. In yet another embodiment, the recombinantvirus comprises the cystic fibrosis transmembrane conductance regulator(CFTR) gene.

Dosage

As will be readily apparent to one skilled in the art, the useful invivo dosage to be administered and the particular mode of administrationwill vary depending upon the age, weight and type of patient beingtreated, the particular pharmaceutical composition employed, and thespecific use for which the pharmaceutical composition is employed. Thedetermination of effective dosage levels, that is the dose levelsnecessary to achieve the desired result, can be accomplished by oneskilled in the art using routine methods as discussed above. Innon-human animal studies, applications of the pharmaceuticalcompositions are commenced at higher dose levels, with the dosage beingdecreased until the desired effect is no longer achieved or adverse sideeffects are reduced or disappear. The dosage for a compound of thepresent invention can range broadly depending upon the desired affects,the therapeutic indication, route of administration and purity andactivity of the compound. Typically, human clinical applications ofproducts are commenced at lower dosage levels, with dosage level beingincreased until the desired effect is achieved. Alternatively,acceptable in vitro studies can be used to establish useful doses androutes of administration of the test compound. Typically, dosages can bebetween about 1 ng/kg and about 10 mg/kg, preferably between about 10ng/kg and about 1 mg/kg, and more preferably between about 100 ng/kg andabout 100 micrograms/kg.

The exact formulation, route of administration and dosage can be chosenby the individual physician in view of the patient's condition (see,Fingle et al., in The Pharmacological Basis of Therapeutics (1975)). Itshould be noted that the attending physician would know how to and whento terminate, interrupt or adjust administration due to toxicity, organdysfunction or other adverse effects. Conversely, the attendingphysician would also know to adjust treatment to higher levels if theclinical response were not adequate. The magnitude of an administrateddoes in the management of the disorder of interest will vary with theseverity of the condition to be treated and to the route ofadministration. The severity of the condition may, for example, beevaluated, in part, by standard prognostic evaluation methods. Further,the dose and perhaps dose frequency, will also vary according to theage, body weight and response of the individual patient, including thosefor veterinary applications.

In some preferred regimens, appropriate dosages are administered to eachpatient by either inhaler, nasal spray, or by topical application. Itwill be understood, however, that the specific dose level and frequencyof dosage for any particular patient may be varied and will depend upona variety of factors including the activity of the specific salt orother form employed, the metabolic stability and length of action ofthat compound, the age, body weight, general health, sex, diet, mode andtime of administration, rate of excretion, drug combination, theseverity of the particular condition, and the host undergoing therapy.

EXAMPLES Example 1 Synthesizing Aprotinin Genes, Purifying and TestingAprotinin Fusion Proteins Introduction

Influenza viral protein hemagglutinin (HA) is the major influenzaenvelope protein. It plays an essential role in viral infection. Theimportance of HA is evidenced by the fact that it is the major targetfor protective neutralizing antibodies produced by the host immuneresponse (Hayden, F G. (1996) In Antiviral drug resistance (ed. D. D.Richman), pp. 59-77. Chichester, UK: John Wiley & Sons Ltd.). It is nowclear that HA has two different functions in viral infection. First, HAis responsible for the attachment of the virus to sialic acid cellreceptors. Second, HA mediates viral entry into target cells bytriggering fusion of the viral envelope with cellular membranes.

HA is synthesized as a precursor protein, HA0, which is transferredthrough the Golgi apparatus to the cell surface as a trimeric molecularcomplex. HA0 is further cleaved to generate the C terminus HA1 (residue328 of HA0) and the N terminus of HA2. It is generally believed that thecleavage occurs at the cell surface or on released viruses. The cleavageof HA0 into HA1/HA2 is not required for HA binding to a sialic acidreceptor; however, it is essential for viral infectivity (Klenk, H D andRott, R. (1988) Adv Vin Res. 34:247-281; Kido, H, Niwa, Y, Beppu, Y. andTowatari, T. (1996) Advan Enzyme Regul 36:325-347; Skehel, J J andWiley, D C. (2000) Annu Rev Biochem 69:531-569).

Sensitivity of HA0 to host proteases is determined by the proteolyticsite in the external loop of HA0 molecule. The proteolytic site maycontain either a single Arg or Lys residue (monobasic cleavage site) orseveral Lys and/or Arg residues in R-X-K/R-R motif (multibasic cleavagesite). Only the influenza A virus subtypes H5 and H7 have HA proteinscarrying the multibasic cleavage site. All other influenza A, B and Cviruses contain HA proteins having the monobasic cleavage site.Influenza A viruses having multibasic cleavage sites are more virulentand induce systemic infection in hosts whereas viruses with a monobasicHA site initiate infection only in the respiratory tract in mammals orin the respiratory and enteric tracts in avian species (Klenk, H D andGarten W. 1994. Trend Micro 2:39-43 for review). Fortunately, humaninfection by the highly virulent avian influenza A H5 and H7 subtypes,which carry the multibasic cleavage site, has so far only occurred in ahandful of cases discovered mostly in Hong Kong. The vast majority ofinfluenza infections are caused by viruses with HA proteins are cleavedat the monobasic cleavage site.

Influenza virus HA subtypes 5 and 7 that contain multibasic cleavagesites are activated by furin, a member of the subtilisin-likeendoproteases, or the pre-protein convertase family. Furin cleaves thevirus intracellularly and is ubiquitously present in many cell types,allowing the virulent, systemic infection seen with such viruses (Klenk,H D and Garten W. 1994. Trend Micro 2:39-43; Nakayama, K. 1997. Biochem327:625-635). All other influenza viruses, which have HAs with monobasiccleavage sites, are activated by secreted, trypsin-like serineproteases. Enzymes that have been implicated in influenza virusactivation include: plasmin (Lazarowitz S G, Goldberg A R and Choppin PW. 1973. Virology 56:172-180), mini-plasmin (Murakami M, Towatari T,Ohuchi M, Shiota M, Akao M, Okumura Y, Parry M A and Kido H. (2001) EurJ Biochem 268: 2847-2855), tryptase Clara (Kido H, Chen Y and MurakamiM. (1999) In B. Dunn (ed.), Proteases of infectious agents. p. 205-21′7,Academic Press, New York, N.Y.), kallikrein, urokinase, thrombin(Scheiblauer H, Reinacher M, Tashiro M and Rott R. (1992) J Infec Dis166:783-791), blood clotting factor Xa (Gotoh B, Ogasawara T, Toyoda T,Inocencio N, Hamaguchi M and Nagai Y. (1990) EMBO J 9:4189-4195),acrosin (Garten W, Bosch F X, Linder D, Rott R and Klenk H D. (1981)Virology 115:361-374.), proteases from human respiratory lavage(Barbey-Morel C L, Oeltmann T N, Edwards K M and Wright P F. (1987) JInfect Dis 155:667-672) and bacterial proteases from Staphylococcusaureus (Tashiro M, Ciborowski P, Reinacher M, Pulverer G, Klenk H D andRott R. (1987) Virology 157:421-430) and Pseudomonas aeruginosa (CallanR J, Hartmann F A, West S E and Hinshaw V S. (1997) J Virol71:7579-7585). Activation of influenza viruses by host serine proteasesis generally considered to occur extracellularly either at the plasmamembrane or after virus release from the cell.

Aprotinin, also called Trasylol, or bovine pancreatic trypsin inhibitor(BPTI) is a polypeptide having 58 amino acids. It belongs to the familyof Kunitz-type inhibitors and competitively inhibits a wide spectrum ofserine proteases, including trypsin, chymotrypsin, plasmin and plasmakallikrein. Aprotinin has long been used as a human therapeutics, suchas treatment of pancreatitis, various states of shock syndrome,hyperfibrinolytic haemorrhage and myocardial infarction. It is also usedin open-heart surgery, including cardiopulmonary bypass operations, toreduce blood loss (Fritz H and Wunderer G. (1983) Arzneim-Forsch33:479-494).

The safety of aprotinin in human has been well documented through yearsof clinical applications. In addition, aprotinin is apparently a veryweak immunogen as aprotinin-specific antibodies have not been observedin human sera so far (Fritz H and Wunderer G. (1983) Arzneim-Forsch33:479-494). Another desired feature of aprotinin as a drug candidate isits superb stability. It can be kept at room temperature for at least 18months without any loss of activity (Fritz H and Wunderer G. (1983)Arzneim-Forsch 33:479-494).

To achieve significant viral inhibition in animal studies that have beenperformed, aprotinin was administered at high doses. For example, 280micrograms to 840 micrograms per day of aprotinin was injectedintraperitoneally into each mouse for 6 days (Zhirnov O P, Ovcharenko AV and Bukrinskaya A G. (1984) J Gen Virol 65:191-196); a lower dosagewas required for aerosol inhalation, still, each mouse was given 63-126micrograms per day for 6 days (Ovcharenko A V and Zhirnov O P. (1994)Antiviral Res 23:107-118). A very high dose of aprotinin would berequired in human based on extrapolation from the mouse data. Thereforeto achieve better efficacy in human, the potency of aprotinin moleculeneeds to be significantly improved.

Aprotinin functions by competitively inhibiting serine proteases thatare mostly on the surface of host respiratory epithelial cells. Localconcentration of aprotinin in the vicinity of host proteases istherefore the key factor determining competitive advantage of aprotinin.We use two approaches that work synergistically to boost competitiveadvantage of aprotinin on the surface of respiratory epithelium.

First, the avidity (functional affinity) of aprotinin is increased bymaking multivalent aprotinin fusion proteins consisting of two, three,or more aprotinin proteins connected via linkers. Such a molecule isable to bind to membrane proteases in a multivalent fashion, which hassignificant kinetic advantage over the aprotinin monomer. Monomericaprotinin binds to bovine trypsin very tightly with dissociationconstant (Ki) being 6.0×10⁻¹⁴ mol/l. However, its affinity compared toother proteases, such as chymotrypsin, plasmin and Kallikrein, whichhave been implicated in activation of influenza viruses, is much lowerwith Ki being at the level of 10⁻⁸ to 10⁻⁹ mol/l (Fritz H and WundererG. (1983) Arzneim-Forsch 33:479-494). Multimerization can increaseaprotinin's affinity to these proteases exponentially.

Second, we fuse aprotinin with a respiratory epithelium-anchoringdomain. The anchoring domain localizes aprotinin to the proximity ofhost membrane-associated proteases and maintains a high localconcentration of aprotinin on epithelial surface. The anchoring domainalso increases retention time of the drug on the respiratory epithelium.

Cloning

Aprotinin is a single chain polypeptide having 58 amino acid residuesand 3 intra-chain disulfide bonds (SEQ ID NO:1). The amino acid sequenceof aprotinin is shown in FIG. 1. Genes encoding aprotinin and aprotininfusion proteins are synthesized by PCR using overlappingoligonucleotides with codons optimized for E. Coli expression astemplates. The PCR products are cloned into pCR2.1-TOPO vector(Invitrogen). After sequencing, the genes are subcloned into anexpression vector pQE (Qiagen). The vector carries a purification tag,Hisx6, to allow easy purification of the recombinant proteins. Theconstructs are used to transform E. Coli. The transformed cells grown inLB-ampicillin medium to mid-log phase are induced by IPTG according tostandard protocols. Cells are pelleted and lysed inphosphate-buffered-saline (PBS) by sonication. The enzymes, which haveHis₆ purification tag, are purified using a nickel column (Qiagen).

The following aprotinin fusion proteins are made:

-   1. Dimeric and trimeric aprotinin. Two or three aprotinin genes are    linked via a flexible linker as the following constructs:    -   Aprotinin-(GGGGS (SEQ ID NO:10))n (n=3, 4 or 5)-Aprotinin; and    -   Aprotinin-(GGGGS(SEQ ID NO:10))n (n=3, 4 or        5)-Aprotinin-(GGGGS(SEQ ID NO:10))n (n=3, 4 or 5)-Aprotinin        The length of the linker sequence may determine        three-dimensional flexibility of the multimeric aprotinin and        thereby influence functional affinity of the molecule. Therefore        constructs having linkers with various lengths are made.

Fully functional recombinant monomeric aprotinin has been produced in E.Coli (Auerswald E A, Horlein D, Reinhardt G, Schroder W and Schnabel E.(1988). Biol Chem Hoppe-Seyler Vol 369, Suppl., pp 27-35). We thereforeexpect proper folding of multivalent aprotinin proteins in E. colicells. Besides expressing protein in various common E. Coli cellstrains, such as BL21, JM83, etc, the multivalent aprotinin proteins arealso expressed in Origami™ cells (Novagen, Bad Soden, Germany). TheOrigami™ cell strain does not have thioredoxin and glutathione reductaseand thus has an oxidizing cytoplasm. This cell strain has been used tosuccessfully express a number of proteins that contain disulfide bonds(Bessette P H, Aslund F, Beckwith J and Georgiou G. (1999) Pro Natl AcadSci USA 96:13703-13708; Venturi M, Seifert C and Hunte C. (2001) J MolBiol 315:1-8).

-   2. The epithelium cell-anchoring aprotinin. An epithelium    cell-anchoring sequence is fused with aprotinin. The    epithelium-anchoring sequence can be any peptide or polypeptide    sequence that has affinity towards the surface of epithelial cells.    We have selected three human GAG-binding sequences: PF4 (aa 47-70;    SEQ ID NO: 2), IL-8 (aa 46-72; SEQ ID NO: 3), and AT III (aa    118-151; SEQ ID NO: 4) (FIG. 2). These sequences bind to    heparin/heparan sulfate with nanomolar-level affinities (Table 1).    Heparin/Heparan Sulfate are ubiquitously present on the respiratory    epithelium. In separate constructs, the GAG-binding sequences are    fused with the aprotinin gene on the N terminus and on the C    terminus via a generic linker sequence GGGGS as the following    constructs:    -   (GAG domain-GGGGS(SEQ ID NO:10)-Aprotinin); and    -   (Aprotinin-GGGGS(SEQ ID NO:10)-GAG domain)

TABLE 1 Affinities to Heparin Protein Kd nM (ref) PF4 27 (44) IL-8 <5(43) ATIII 11 (42) ApoE 620 (45) 

Photometric Trypsin Inhibition Assay

The trypsin inhibition activity of aprotinin and aprotinin fusionproteins is measured by a photometric assay described previously indetail (Fritz H and Wunderer G. (1983) Arzneim-Forsch 33:479-494).Briefly, in this assay aprotinin inhibits the trypsin-catalyzedhydrolysis of Na-benzoyl-L-arginine-p-nitroanilide (BzArgpNA or L-BAPA)(Sigma), which is followed photometrically at 405 nm. One trypsin unit(U_(BAPA)) corresponds to the hydrolysis of 1 micromole substrate permin. One inhibitor unit (IU_(BAPA)) decreases the activity of twotrypsin units by 50%, which corresponds arithmetically to the inhibitionof 1 U_(BAPA) of trypsin. The specific activity of aprotinin is given inIU_(BAPA)/mg polypeptide.

Surface Plasmon Resonance Assay

The affinities of dimeric and trimeric aprotinin with various linkersare compared against the monomeric aprotinin using surface plasmonresonance assay, or BIAcore analysis (BIAcore, Piscataway, N.J.) withhuman plasmin as the target. Similarly, BIAcore assay with heparin asthe target is used to analyze affinity between GAG binding aprotininfusion proteins and heparin.

When plasmin is used as the target, purified human plasmin (Sigma) isimmobilized on the CM5 chip according manufacturer's instructions(BIAcore, Piscataway, N.J.). When heparin is the target, biotinylatedalbumin and albumin-heparin (Sigma) are captured on astreptavidin-coated BIAcore SA chip as described previously (Xiang Y andMoss B. (2003) J Virol 77:2623-2630).

Example 2 Establishing Improved Tissue Culture Models for Studies onInfluenza Virus Infection Stocks of Influenza Viruses

Influenza viral strains are obtained from ATCC and the repository at St.Jude Children's Research Hospital. All experiments involving influenzaviruses are conducted at Bio-safety level II.

Viruses are propagated by injection into the allantoic cavity ofnine-day-old chicken embryos as described (Zhirnov O P, Ovcharenko A Vand Bukrinskaya A G. (1985) J Gen Virol 66:1633-1638). Alternatively,viral stocks are grown on Madin-Darby canine kidney (MDCK) cells inminimal essential medium (MEM) supplemented with 0.3% bovine serumalbumin and 0.5 micrograms of trypsin per ml. After incubating for 48 to72 hours, the culture medium is clarified by low speed centrifugation.Viral particles are pelleted by ultracentrifugation through a 25%sucrose cushion. Purified viruses are suspended in 50% glycerol-0.1MTris buffer (pH 7.3) and stored at −20° C.

Plaque Assays

Infectivity and titer of the viral stocks are determined by two kinds ofplaque assays, a conventional one and a modified one (Tobita, K,Sugiura, A, Enomoto, C. and Furuyama, M. (1975) Med Microbiol Immnuol162:9-14; Zhirnov O P, Ovcharenko A V and Bukrinskaya A G. (1982) ArchVirol 71:177-183). The conventional plaque assay is routinely used as avirus titration method. It requires exogenous trypsin in agar overlayadded immediately after virus infection to MDCK monolayers (Tobita, K,Sugiura, A, Enomoto, C. and Furuyama, M. (1975) Med Microbiol Immnuol162:9-14). This method artificially increases infectivity of the viralstocks being tested by activating all the viral particles that haveuncleaved HA.

Zhirnov et. al. designed a modified plaque assay consisting of a doubleagar overlay, with trypsin being included in the second layer which isadded 24 hours after infection (Zhirnov O P, Ovcharenko A V andBukrinskaya A G. (1982) Arch Virol 71:177-183). Three days afterinfection, cells are fixed with a 10% formaldehyde solution, agaroselayers are removed, fixed cells are stained with hematoxylin-eosinsolution and plaques are counted. The modified plaque assay allowsaccurate determination of the real infectivity of viral stocks thatcontain both cleaved and uncleaved HA. Combining results from bothconventional and modified plaque assays, one can distinguish virusescontaining cleaved or uncleaved HA and correlate infectivity of viralstocks with the status of HA cleavage.

Human Cell Culture Models

1. Short-term culture of primary human epithelial cells. Conventional invitro influenza virus infection is mostly carried out in MDCK cells withexogenous trypsin added to the culture medium. This is far from beingphysiological and is inappropriate for the work proposed here becausetrypsin is not the protease that activate influenza viruses in vivo.Very limited numbers of in vitro tissue culture models that are able tosupport the growth of influenza virus without an exogenous protease havebeen reported so far, those being primary cultures with primate cells ofrenal origin, cells lining the allantoic and amniotic cavities ofembryonated eggs, fetal tracheal ring organ cultures and primary humanadenoid epithelial cells (Endo Y, Carroll K N, Ikizler M R and Wright PF. (1996) J Virol 70:2055-2058). Among these, the latest work withprimary human adenoid epithelial cells is the closest mimic of humanconditions. In this case, Endo et. al. (Endo Y, Carroll K N, Ikizler M Rand Wright P F. (1996) J Virol 70:2055-2058) isolated epithelial cellsfrom surgical samples of human adenoids, and cultured the epithelialcells on a collagen matrix (Vitrogen 100, Celtrix Laboratories, PaloAlto, Calif.) in Transwell inserts (Costar, Cambridge, Mass.). Cellswere maintained in 50% Ham's F12 and 50% Eagles minimal essential mediawith supplements of growth factors and trace elements. The cells reachedconfluency in 10 to 14 days, remaining largely as a monolayer but withdiscrete patches of ciliated cells, which maintained regular ciliaryactivity for 1 to 3 weeks after reaching confluency. In this system,influenza A virus grew to a titer of 10⁶ PFU/ml with a multiplicity ofinfection of 0.001 (Endo Y, Carroll K N, Ikizler M R and Wright P F.(1996) J Virol 70:2055-2058). Progressive cytopathogenic effects werealso present during infection. The biggest drawback of this system isthat it requires fresh human adenoid tissue.

To solve this problem, primary human adenoid epithelial cells arereplaced with primary human airway epithelial cells that arecommercially available (Cambrex), and the cells are grown under the sameconditions. Such short-term culture of primary human airway epithelialcells is relatively quick to establish and is useful as the first-lineexperimental model for most of the in vitro infection and antiviralexperiments.

2. Well-differentiated human airway epithelium (WD-HAE). In order tobest mimic the in vivo condition of human airway, the model ofwell-differentiated human airway epithelium (WD-HAE) is used. WD-HAE isstratified epithelium that has all the differentiated cells of thenormal human airway epithelium, including functional ciliated cells andmucus secreting cells. Therefore, in this model system influenza virusesare most likely to be activated by host proteases that arephysiologically relevant. Although WD-HAE has been widely used to studyrespiratory viral infections, such as respiratory syncytial virus (RSV)(Zhang L, Peeples M E, Boucher R C, Collins P L and Pickles R J. (2002)J Virol 76:5654-5666) measles virus (Sinn P L, Williams G, VongpunsawadS, Cattaneo R and McCray P B. (2002) J Virol 76:2403-2409, or humanrhinovirus, it has not previously been used to study influenza viruses.

A detailed protocol of WD-HAE has been described previously (Krunkosky TM, Fischer B M, Martin L D, Jones N, Akley N J and Adler K B. (2000) AmJ Respir Cell Mol Biol 22:685-692). Briefly, commercial primary humanbronchial epithelial cells (Cambrex) are cultured on Transwell-clearculture inserts (Costar) that are thin-coated with rat-tail collagen I.Cells are cultured submerged for the first 5 to 7 days in mediumcontaining a 1:1 mixture of bronchial epithelial cell growth medium(BEGM) (Cambrex) and DMEM with high glucose with supplement of growthfactors (Krunkosky T M, Fischer B M, Martin L D, Jones N, Akley N J andAdler K B. (2000) Am J Respir Cell Mol Biol 22:685-692). When culturesare 70% confluent (days 5 to 7), the air-liquid interface is created byremoving the apical medium and exposing cells only to medium on theirbasal surface. Cells are cultured for additional 14 days in air-liquidinterphase, for a total of 21 days in culture, and are then ready forexperiments. The differentiated epithelium can be maintained in vitrofor weeks.

Epithelial morphology and degree of differentiation is documented byroutine histology (Endo Y, Carroll K N, Ikizler M R and Wright P F.(1996) J Virol 70:2055-2058). Briefly, following fixation with 10%buffered formalin, the epithelial cells are embedded in paraffin,sectioned and stained with hematoxylin and eosin, and with periodicacid-Schiff stain for mucus secreting cells.

Influenza infection is carried out in the above two model systems byadding 0.001 to 1 MOI of viruses to the differentiated cells. The titerand infectivity of viruses in the supernatant are followed over a periodof 3 to 7 days. The level of influenza viral amplification and theinfectivity of influenza viruses are evaluated using conventional andmodified plaque assays.

Example 3 Comparing Functions of the Aprotinin Fusion Proteins In VitroAnti-Viral Effects of Aprotinin Fusion Proteins

1. Pre-infection treatment. Aprotinin fusion proteins are added toprimary human cell cultures at various concentrations and allowed toincubate with the cells for 1 hour. The cells are washed with freshmedium and immediately inoculated with influenza viruses at MOI 0.01to 1. Cells are washed again after 1 hour and cultured for 3 to 5 days.Titer and infectivity of viruses in the supernatant are measured atvarious time points by two plaque assays. The cytopathic effect causedby viral infection is evaluated by staining viable cells with crystalviolet and quantifying by measuring absorption at 570 nm at the end ofthe experiment. The percentage of cell protection by aprotinin fusionproteins is calculated by 100×{(aprotinin treated sample-untreatedinfected sample)/(uninfected control-untreated infected sample)}. Thedrug efficacy for cell protection is described by its EffectiveConcentration that achieves 50% of the cell protection (EC₅₀). Since HAactivation only occurs to newly released viral particles, the firstround of viral infection occurs normally and viral titer rises in thefirst 24 hours after infection. However, starting from the second round,infectivity of viruses drops and viral titer gradually decreases asresult of aprotinin treatment. Results from this experimentdifferentiate various types of different aprotinin fusion proteins bytheir efficacies in a single prophylactic treatment.

Alternatively, timing of initial viral inoculation is altered fromimmediately after aprotinin treatment to 2-24 hours post treatment.Viral titer, infectivity and cytopathic effect are measured for 3 to 5day after infection as described above. Results from these experimentsdistinguish various aprotinin fusion proteins by the lengths of theeffective window after a single prophylactic treatment.

2. Post-infection Treatment. For multi-dose treatment, cells are firstinfected by viral inoculations at 0.001 to 0.1 MOI for 1 hour. Variousconcentrations of aprotinin fusion proteins are added immediatelyafterwards, additional treatments are applied at 8-hour intervals duringthe first 48 hours post infection. Cells are cultured until day 7 postinfection. Viral titer and infectivity in the media are followed duringthe whole process. Cytopathic effect is evaluated at the end of theexperiment.

For single dose treatment, cells are first infected by viralinoculations at 0.001 to 0.1 MOI for 1 hour. Treatments of aprotininfusion proteins at various concentrations are applied at different timepoints during the first 48 hours after infection, but each cell sampleonly receives one treatment during the whole experiment. Cells arecultured until day 7 post infection. Viral titer and infectivity in themedia are followed during the whole process. Cytopathic effect isevaluated at the end of the experiment. Results from these experimentsdistinguish different types of aprotinin fusion proteins for theirtherapeutic potency.

Inhibition of HA Cleavage by Aprotinin Fusion Proteins

To demonstrate that aprotinin fusion proteins inhibit influenza viralinfection by inhibiting cleavage of influenza HA protein, a humanprimary epithelial cell culture is infected with influenza virus at MOIof 1. Aprotinin fusion proteins are added to the culture either rightbefore viral inoculation or immediately after the viral infection. At6.5 hour post infection, the culture is incubated for 1 hour in MEMlacking cold methionine and containing ³⁵S-labeled methionine (Amersham)at a concentration of 100 microCi/ml (pulse). Thereafter, the cells arewashed twice with MEM containing a 10-fold concentration of coldmethionine and incubated in MEM for additional 3 hours (chase). Afterlabeling, cells are dissolved in radioimmunoprecipitation assay (RIPA)buffer, HA is precipitated by anti-serum against the particular strainof virus used for infection (anti-influenza sera can be obtained fromATCC and Center of Disease Control and Prevention), and theimmunocomplex is then purified by protein G-Sepharose (Amersham).Samples are fractionated by SDS-PAGE followed by autoradiography. Insamples untreated by aprotinin fusion proteins, HA1 and HA2 are expectedto be the predominant HA species; while in aprotinin treated samples,HA0 is expected to be the major type of HA present.

Example 4 Synthesizing Genes of Five Sialidases, Expressing andPurifying the Sialidase Proteins Introduction

Influenza viruses belong to the orthomyxoviridae family of RNA viruses.Both type A and type B viruses have 8 segmented negative-strand RNAgenomes enclosed in a lipid envelope derived from the host cell. Theviral envelope is covered with spikes that are composed of threeproteins: hemagglutinin (HA), that attaches virus to host cell receptorsand mediates fusion of viral and cellular membranes; neuraminidase (NA),which facilitates the release of the new viruses from the host cell; anda small number of M2 proteins that serve as ion channels. For InfluenzaA virus, HA and NA both undergo antigenic drift and antigenic shift, theviral subtypes are distinguished by serologic differences between theirHA and NA proteins. There are total 15 types of HA (H1-H15) and 9 typesof NA (N1-N9), but only three HA (H1-H3) and two NA (N1 and N2) havebeen found in human Influenza A virus so far (Granoff, A. & Webster, R.G., ed. Encyclopedia of Virology, 2^(nd) Edition, Vol 2). In contrast toInfluenza A virus, no distinct antigenic subtypes are recognized forInfluenza virus B.

While Influenza B virus circulates only in humans, Influenza A virus canbe isolated from a whole host of animals, such as pigs, horses,chickens, ducks and other kinds of birds, which accounts for geneticreassortment of Influenza A virus that results in antigenic shift. Wildaquatic birds are considered to be the primordial reservoir of allinfluenza viruses for avian and mammalian species. There is extensiveevidence for transmission of the virus between aquatic birds and otherspecies including pigs and horses and indirect transmission to humansthrough pigs. Direct transmission from pigs or chickens to humans hasalso been documented (Ito, T. (2000) Microbiol Immunol 44 (6):423-430).

The host cell receptor for influenza viruses is the cell surface sialicacid. Sialic acids are α-keto acids with 9-carbon backbones that areusually found at the outermost positions of the oligosaccharide chainsthat are attached to glycoproteins and glycolipids. One of the majortypes of sialic acids is N-acetylneuraminic acid (Neu5Ac), which is thebiosynthetic precursor for most of the other types. Two major linkagesbetween Neu5Ac and the penultimate galactose residues of carbohydrateside chains are found in nature, Neu5Ac α(2,3)-Gal and Neu5Acα(2,6)-Gal. Both Neu5Ac α(2,3)-Gal and Neu5Ac α(2,6)-Gal molecules canbe recognized by Influenza A virus as the receptor (Schauer, R. (1982)Adv. Carbohydrate Chem & Biochem 40:131-235), while human viruses seemto prefer Neu5Ac α(2,6)-Gal, avian and equine viruses predominantlyrecognize Neu5Ac α(2,3)-Gal (Ito, T. (2000) Microbiol Immunol 44(6):423-430).

Infections by influenza type A and B viruses are typically initiated atthe mucosal surface of the upper respiratory tract. Viral replication isprimarily limited to the upper respiratory tract but can extend to thelower respiratory tract and causes bronchopneumonia that can be fatal.The risk of death is one per 10,000 infections, but is significantlygreater for high-risk groups with pre-existing cardiopulmonaryconditions and for immunologically naïve individuals during a pandemic.

A therapeutic compound comprising a sialidase that can effectivelydegrade both receptor sialic acids, Neu5Ac α(2,6)-Gal and Neu5Acα(2,3)-Gal, can confer protection against the broadest range ofinfluenza viruses, including animal viruses. It can also remaineffective as the viral strains change yearly. Because sialidase targetsthe host cell rather than virus and acts at the “choking point” in aviral life cycle, generation of resistant virus is improbable.Protein-bound sialic acid turns over homogeneously on cell surface withhalf-life of 33 hours (Kreisel, W, Volk, B A, Buchsel, R. and Reutter,W. (1980) Proc Natl Acad Sci USA 77:1828-1831). Therefore we estimatethat once-a-day or twice-a-day administration of a sialidase wouldconfer sufficient protection against influenza.

Sialidases are found in higher eukaryotes, as well as in some mostlypathogenic microbes, including viruses, bacteria and protozoans. Viraland bacterial sialidases have been well characterized, and thethree-dimensional structures of some of them have been determined(Crennell, S J, Garman, E, Layer, G, Vimr, E. and Taylor, G. (1994)Structure 2:535-544; Janakiraman, M N, White, C L, Layer, W G, Air, G Mand Luo, M. (1994) Biochemistry 33:8172-8179; Pshezhetsky, A, Richard,C, Michaud, L, Igdoura, S, Wang, S, Elsliger, M, Qu, J, Leclerc, D,Gravel, R, Dallaire, L. and Potier, M. (1997) Nature Genet. 15:316-320). Several human sialidases have also been cloned in the recentyears (Milner, C M, Smith, S V, Carrillo M B, Taylor, G L, Hollinshead,M and Campbell, R D. (1997) J Bio Chem 272:4549-4558; Monti, E, Preti,A, Nesti, C, Ballabio, A and Borsani G. 1999. Glycobiol 9:1313-1321;Wada, T, Yoshikawa, Y, Tokuyama, S, Kuwabara, M, Akita, H. and Miyagi,T. (1999) Biochem Biophy Res Communi 261:21-27; Monti, E, Bassi, M T,Papini, N, Riboni, M, Manzoni, M, Veneranodo, B, Croci, G, Preti, A,Ballabio, A, Tettamanti, G. and Borsani, G. (2000) Bichem J349:343-351). All the sialidases characterized share a four amino acidmotif in the amino terminal portion followed by the Asp box motif whichis repeated three to five times depending on the protein. (Monti, E,Bassi, M T, Papini, N, Riboni, M, Manzoni, M, Veneranodo, B, Croci, G,Preti, A, Ballabio, A, Tettamanti, G. and Borsani, G. (2000) Bichem J349:343-351; Copley, R R, Russell, R B and Ponting, C P. (2001) ProteinSci 10:285-292). While the overall amino acid identity of the sialidasesuperfamily is relatively low at about 20-30%, the overall fold of themolecules, especially the catalytic amino acids, are remarkably similar(Wada, T, Yoshikawa, Y, Tokuyama, S, Kuwabara, M, Akita, H. and Miyagi,T. (1999) Biochem Biophy Res Communi 261:21-27; Monti, E, Bassi, M T,Papini, N, Riboni, M, Manzoni, M, Veneranodo, B, Croci, G, Preti, A,Ballabio, A, Tettamanti, G. and Borsani, G. (2000) Bichem J 349:343-351;Copley, R R, Russell, R B and Ponting, C P. (2001) Protein Sci10:285-292).

The sialidases are generally divided into two families: “small”sialidases have molecular weight of about 42 kDa and do not requiredivalent metal ion for maximal activity; “large” sialidases havemolecular weight above 65 kDa and may require divalent metal ion foractivity (Wada, T, Yoshikawa, Y, Tokuyama, S, Kuwabara, M, Akita, H. andMiyagi, T. (1999) Biochem Biophy Res Communi 261:21-27; Monti, E, Bassi,M T, Papini, N, Riboni, M, Manzoni, M, Veneranodo, B, Croci, G, Preti,A, Ballabio, A, Tettamanti, G. and Borsani, G. (2000) Bichem J349:343-351; Copley, R R, Russell, R B and Ponting, C P. (2001) ProteinSci 10:285-292).

Over fifteen sialidase proteins have been purified and they vary greatlyfrom one another in substrate specificities and enzymatic kinetics. Toconfer a broad-spectrum protection against influenza viruses, asialidase needs to effectively degrade sialic acid in both α(2,6)-Galand α(2,3)-Gal linkages and in the context of glycoproteins and someglycolipids. Viral sialidases, such as those from influenza A virus,fowl plague virus and Newcastle disease virus, are generally specificfor Neu5Ac α(2,3)-Gal and only degrade Neu5Ac α(2,6)-Gal veryinefficiently. Small bacterial sialidases generally react poorly tosialic acid in the context of glycoproteins and glycolipids. Bycontrast, large bacterial sialidases can effectively cleave sialic acidin both (α,2-6) linkage and (α,2-3) linkage in the context of mostnatural substrates (FIG. 4; Vimr, D R. (1994) Trends Microbiol 2:271-277; Drzeniek, R. (1973) Histochem J 5:271-290; Roggentin, P,Kleineidam, R G and Schauer, R. (1995) Biol Chem Hoppe-Seyler376:569-575; Roggentin, P, Schauer, R, Hoyer, L L and Vimr, E R. (1993)Mol Microb 9:915-921). Because of their broad substrate specificities,large bacterial sialidases are better candidates.

Among the large bacterial sialidases with known substrate specificityshown in FIG. 4, Vibrio cholerae sialidase requires Ca2+ for activitymaking it less preferred. More preferred sialidases include the 71 kDaenzyme from Clostridium perfringens, the 113 kDa enzyme from Actinomycesviscosus and sialidase of Arthrobacter ureafaciens. A third sialidase,the 68 kDa enzyme from Micromonospora viridifaciens, has been known todestroy influenza viral receptor (Air, G M and Layer, W G. (1995)Virology 211:278-284), and is also a candidate.

These enzymes have high specific activity (600 U/mg protein for C.perfringens (Corfield, A P, Veh, R W, Wember, M, Michalski, J C andSchauer, R. (1981) Bichem J 197:293-299) and 680 U/mg protein for A.viscosus (Teufel, M, Roggentin, P. and Schauer, R. (1989) Biol ChemHoppe Seyler 370:435-443)), are fully active without divalent metaliron, and have been cloned and purified as recombinant proteins from E.coli (Roggentin, P, Kleineidam, R G and Schauer, R. (1995) Biol ChemHoppe-Seyler 376:569-575, Teufel, M, Roggentin, P. and Schauer, R.(1989) Biol Chem Hoppe Seyler 370:435-443, Sakurada, K, Ohta, T. andHasegawa, M. (1992) J Bacteriol 174: 6896-6903). In addition, C.perfringens is stable in solution at 2-8° C. for several weeks, and at4° C. in the presence of albumin for more than two years (Wang, F Z,Akula, S M, Pramod, N P, Zeng, L. and Chandran, B. (2001) J Virol75:7517-27). A. viscosus is labile towards freezing and thawing, but isstable at 4° C. in 0.1 M acetate buffer, pH 5 (Teufel, M, Roggentin, P.and Schauer, R. (1989) Biol Chem Hoppe Seyler 370:435-443).

Although the chances of inducing immune reactions using bacterialsialidases is very low because the proteins will be used topically inthe upper respiratory tract and will not be absorbed systemically, ahuman enzyme would be more desirable for long-term use in humansubjects.

Four sialidase genes have been cloned from human so far:NEU1/G9/lysosomal sialidase (Pshezhetsky, A, Richard, C, Michaud, L,Igdoura, S, Wang, S, Elsliger, M, Qu, J, Leclerc, D, Gravel, R,Dallaire, L. and Potier, M. (1997) Nature Genet. 15: 316-320.

Milner, C M, Smith, S V, Carrillo M B, Taylor, G L, Hollinshead, M andCampbell, R D. (1997). J Bio Chem 272:4549-4558); NEU3, amembrane-associated sialidase isolated from human brain (Wada, T,Yoshikawa, Y, Tokuyama, S, Kuwabara, M, Akita, H. and Miyagi, T. (1999)Biochem Biophy Res Communi 261:21-27, Monti, E, Bassi, M T, Papini, N,Riboni, M, Manzoni, M, Veneranodo, B, Croci, G, Preti, A, Ballabio, A,Tettamanti, G. and Borsani, G. (2000) Bichem J 349:343-351), NEU2 a 42kDa sialidase expressed in human skeletal muscle at a very low level(Monti, E, Preti, A, Nesti, C, Ballabio, A and Borsani G. (1999)Glycobiol 9:1313-1321), and NEU4 a 497 amino acid protein (GenbankNM080741) expressed in all human tissues examined (Monti, E, Preti, A,Venerando, B. and Borsani, G. (2002) Neurochem Res 27:646-663).

Amino acid sequence comparison reveals NEU2 (SEQ ID NO:8) and NEU4 (SEQID NO:9) are both cytosolic sialidases. 9 out of 12 of the amino acidresidues which form the catalytic site of S. typhimurium sialidase areconserved in both NEU2 and NEU4 (Monti, E, Preti, A, Nesti, C, Ballabio,A and Borsani G. (1999) Glycobiol 9:1313-1321, FIG. 3). In addition,NEU4 also shows a stretch of about 80 amino acid residues (aa 294-373)that appears unique among known mammalian sialidases (Monti, E, Preti,A, Venerando, B. and Borsani, G. (2002) Neurochem Res 27:646-663).Unlike the selected large bacterial sialidases, the substratespecificity of NEU2 and NEU4 is unknown. It will need to be tested ifNEU2 and NEU4 can effectively degrade the influenza virus receptors.

Sialidase Assay

NEU2, NEU4 and M. viridifaciens enzymes will be stored in PBS and 50%glycerol at −20° C. C. perfringens and A. viscosus enzymes are stored in10 mM acetate buffer (pH5) at 4° C. Protein preps are characterized byHPLC and SDS-PAGE electrophoresis. Specific activities and stability ofthe enzymes will be monitored by sialidase assay.

The enzymatic activity of sialidases are determined by fluorimetric2′-(4-methylumbelliferyl)-alpha-D-N-acetylneuraminic acid) (4Mu-NANA)(Sigma) as the substrate. Specifically, reactions are set up induplicate in 0.1M Na citrate/phosphate buffer pH5.6, in the presence of400 micrograms bovine serum albumin, with 0.2 mM 4MU-NANA in a finalvolume of 100 microliters, and incubated at 37° C. for 5-10 min.Reactions are stopped by addition of 1 ml of 0.2 M glycines/NaOH pH10.2.Fluorescence emission is measured on a fluorometer with excitation at365 nm and emission at 445 nm, using 4-methylumbelliferone (4-MU) toobtain a calibration curve.

Example 5 Comparing Functions of the Sialidases In Vitro and SelectingOne Sialidase for Further Studies 1. Stocks of Influenza Viruses

Influenza viral strains are obtained from the ATCC and the repository atSt. Jude Children's Research Hospital. Viral stocks are grown onMadin-Darby canine kidney (MDCK) cells in minimal essential medium (MEM)supplemented with 0.3% bovine serum albumin and 0.5 micrograms oftrypsin per ml. After incubating for 48 to 72 hours, the culture mediumis clarified by low speed centrifugation. Viral particles are pelletedby ultracentrifugation through a 25% sucrose cushion. Purified virusesare suspended in 50% glycerol-0.1M Tris buffer (pH 7.3) and stored at−20° C. Viral titer is determined by plaque assay (Tobita, K, Sugiura,A, Enomoto, C. and Furuyama, M. (1975) Med Microbiol Immnuol 162: 9-14),or TCID₅₀, which is the dose of virus required to infect 50% of the MDCKcells.

Selected human and animal influenza A strains with specificity towardsNeu5Ac alpha(2,6)-Gal or Neu5Ac alpha(2,3)-Gal and have high affinity tothe receptors (measured by high hemagglutination activity) are chosenfor in vitro tests:

-   -   1. Strains that recognize receptor Neu5Ac alpha(2,6)-Gal include        human isolates A/aichi/2/68, A/Udorn/307/72, A/Prot        Chaimers/1/73 and A/Victoria/3/75, etc. (Connor, R J, Kawaoka,        Y, Webster, R G and Paulson J C. (1994) Virology 205:17-23).    -   2. Strains that have Neu5Ac alpha(2,3)-Gal specificity include        animal isolates A/duckUkraine/1/63, A/duckMemphis/928/74,        A/duckhokk/5/77, A/Eq/Miami/1/63, A/Eq/Ur/1/63, A/Eq/Tokyo/71,        A/Eq/Prague/71, etc (Connor, R J, Kawaoka, Y, Webster, R G and        Paulson J C. (1994) Virology 205:17-23).

2. Hemagglutination Assay

This assay is used to rapidly determine the efficiency of each enzyme todestroy receptors Neu5Ac alpha(2,6)-Gal and Neu5Ac alpha(2,3)-Gal.

Specifically, 6 ml of Chicken red blood cells (SPAFAS Inc., Norwich,Conn.) are diluted in two times the volume of PBS, centrifuge for 5 minat 500×g and re-suspended in PBS of original volume. Sialidases areadded to the chicken erythrocytes at various concentrations and allowedto incubate at room temperature for 30 min. The cells are then washedthree times to remove sialidase proteins, and then are resuspended inPBS to 6 ml. Control cells are incubated with BSA and washed. Variousstrains of influenza virus, which recognize either Neu5Ac alpha(2,6)-Galor Neu5Ac alpha(2,3)-Gal as the receptor as listed above, are preparedin microtiter plates as serial dilutions in PBS (100 microliters) of theoriginal viral stocks. Sialidase-treated or control chicken red bloodcell suspensions (100 microliters of the 0.5% solution prepared above)are added to each well at 4° C. The plates are read after 2 h. Thelowest concentration of virus that causes the blood cell to agglutinateis defined as one hemagglutination unit. We will be looking for enzymesthat effectively abolish hemagglutination by all viral strains.

3. Viral Inhibition Assay

Confluent monolayers of MDCK cells are treated with variousconcentrations of sialidases for 1 h, washed twice with buffer, theninfected with various strains of influenza virus. After incubation for 1hr, the cells are washed again to remove unbound virus. To estimate thedecrease in viral binding sites on cell surface, the cells are overlaidwith agar and incubated at 37° C. The number of plaques in the sialidasetreated cells will be compared against those in control cells.Alternatively, the cells will be cultured in regular medium at 37° C.,and viral titers in the culture media are measured at various timeduring culture as TCID₅₀.

To demonstrate that sialidase treatment can inhibit a pre-existinginfection, MDCK monolayers are first infected with a low titer of virus.After washing off the unbound virus, the cells are then cultured in thepresence of a sialidase. Fresh sialidase is added to cell culture very24 h. Viral titer in the cultured medium is measured over a 72-hourperiod.

4. Cytotoxicity Assay

Primary human bronchial epithelial cells are purchased (Clonetics) andcultured in supplemented minimal medium following manufacture'sinstruction. Sialidases are added to the culture medium at variousconcentrations. Cell growth over a period of 7-10 days will be measured.Cells will also be observed regularly for microscopic cytopathiceffects.

Example 6 Constructing and Testing Sialidase Fusion Proteins 1. Choosinga GAG-Binding Sequence as the Anchoring Domain.

One sialidase is selected for its best overall properties, includinganti-viral activity, toxicity, stability, ease of production, etc. Wewill then genetically link it to a GAG-binding sequence, sub-clone thefusion genes into pQE vector, express and purify the fusion proteinsfrom E. coli.

We have selected six possible human GAG-binding sequences: PF4 (aa47-70) (SEQ ID NO:2), IL-8 (aa 46-72) (SEQ ID NO:3), AT III (aa 118-151)(SEQ ID NO:4), ApoE (aa 132-165) (SEQ ID NO:5), human angio-associatedmigratory cell protein (AAMP) (aa 14-25) (SEQ ID NO:6), and amphiregulin(AR) (aa 125-145) (SEQ ID NO:7) (FIG. 2). These sequences generally bindto heparin with nanomolar-level affinities; however, their affinitiesmay vary from one another by an order of magnitude (Table 1). Since itis not clear which anchoring domain will enable the most effectivefunctioning of the sialidase, all four GAG-binding sequences are fusedwith the sialidase gene either on the N terminus or the C terminus via ageneric linker sequence GGGGS as the following constructs:

-   -   (GAG binding domain-GGGGS(SEQ ID NO:10)-Sialidase); or        (Sialidase-GGGGS(SEQ ID NO:10)-GAG binding domain)

Different fusion proteins are compared by a modified viral inhibitionassay. Specifically, confluent monolayers of MDCK cells are treated withsame amount of each fusion protein for a limited duration, such as 30min. The cells are then washed twice with buffer to remove unboundsialidase fusion proteins, and incubated in culture medium for anadditional 1 hour. Afterwards, strains of influenza virus are added tothe cells for 1 hr and then cells are washed again to remove unboundvirus. Viral titers in the culture media are measured during 72-hcultures as TCID₅₀. The un-fused sialidase protein will be used tocompare against the fusion proteins in this assay. If the results aretoo close to rank all fusion proteins, we will make the assay morestringent by shortening treatment window for the fusion proteins,lowering protein concentrations and increasing the level of viralchallenge.

2. Optimizing the Fusion Protein Construct

After selecting the best fusion protein from the earlier experiments,the construct is further optimized by testing different linker length.In this regard, the following constructs are made:

(Sialidase-(GGGGS(SEQ ID NO:10))n (n=0, 1, 2, 3, or 4)-GAG bindingdomain)The proteins are expressed and purified and compared in the modifiedviral protection assay as described above.

In addition, if earlier data indicate that higher affinity of the fusionprotein towards heparan sulfate brings better potency, we also plan totest if the potency can be further improved by increasing theGAG-binding affinity. This can be achieved by creating a multivalent GAGbinding mechanism in the fusion protein in constructs like these:

(Sialidase-(GGGGS(SEQ ID NO:10))n-HS binding domain-GAG binding domain);or:(GAG binding domain-(GGGGS(SEQ ID NO:10))n-Sialidase-(GGGGS(SEQ IDNO:10))n-GAG binding domain)

The purified fusion proteins are ranked based on their activities in themodified viral protection assay as described above.

3. Cytotoxicity Assay

The effects of the fusion proteins on normal cell growth and morphologyare monitored by culturing primary human bronchial epithelial cells withvarious concentrations of the fusion proteins and following growth curveof the cells and observing any microscopic cytopathic effects.

Example 7 Fusion Proteins Against Other Infectious Microbes

Fusion proteins composed of a functional domain and an anchorage domainare designed for many more different applications. For example, asialidase fusion protein as proposed here can also be used as atherapeutic/prophylactic agent against infections by other viruses andbacteria besides influenza viruses, because many other infectiousmicrobes, such as paramyxoviruses (Wassilewa, L. (1977) Arch Virol54:299-305), coronaviruses (Vlasak, R., Luytjes, W., Spaan, W. andPalese, P. (1988) Proc Natl Acad Sci USA 85:4526-4529), rotaviruses(Fukudome, K., Yoshie, O. and Konno, T. (1989) Virology 172:196-205) andPseudomonas aeruginosa (Ramphal, R. and Pyle, M. (1983) Infect Immun41:339-44) etc, are also known to use sialic acid as cellular receptors.For example, aprotinin fused with a heparin-binding domain can make afusion protein that be used to prevent/treat infection of other virusesbesides influenza that require host serine proteases for activation,such as parainfluenza virus.

Example 8 Cloning Sialidase Catalytic Domain Fusion Proteins

According to the published literature on the large bacterial sialidases,the 51 kDa Arthrobacter ureafaciens sialidase, the 71 kDa sialidase fromClostridium perfringens and the 113 kDa sialidase from Actinomycesviscosus seem to have similar specific activities and broad substratespecificity toward various sialic acid conjugates (Biology of the SialicAcids (1995), 270-273; Corfield et al., Biochem. J., (1981) 197 (2),293-299; Roggentin et al., Biol. Chem. Hoppe Seyler, (1995) 376 (9),569-575; Teufel et al., Biol. Chem. Hoppe Seyler, (1989) 370 (5),435-443). A third sialidase, the 68 kDa enzyme from Micromonosporaviridifaciens, was also known to destroy the influenza viral receptor(Air and Layer, Virology, (1995) 211 (1), 278-284; (1995), 270-273).

A. viscosus is part of the normal flora of human oral cavity andgastrointestinal tract (Sutter, Rev. Infect. Dis., (1984) 6 Suppl 1,S62-S66). Since the sialidase from A. viscosus is normally secreted bythe bacterium hosted on human mucosal surface, it should be tolerated bythe human mucosal immune system. Therefore, it is unlikely that A.viscosus sialidase will be immunogenic when delivered topically to thehuman airway surface. We think that this feature makes A. viscosussialidase a good candidate for a therapeutic agent.

We determined that a fragment of the A. viscosus sialidase, extendingfrom amino acid 274 to amino acid 667, should contain the catalyticdomain (referred to as AvCD) of the sialidase and should be fully activeon it own. We later cloned the AvCD fragment and demonstrated that thisAvCD fragment and other A. viscosus sialidase fragments comprising atleast amino acids 290-666 of the A. viscosus sialidase protein sequence(SEQ ID NO:12), such as the fragment extending from amino acid 274 toamino acid 681, the fragment extending from amino acid 274 to amino acid666, the fragment extending from amino acid 290 to amino acid 666, andthe fragment extending from amino acid 290 to amino acid 681, havesialidase activity.

The complete sequence of the A. viscosus sialidase protein and gene areset forth in SEQ ID NOS: 11 and 12, respectively. Based on homology withsialidases with known 3D structures (M. viridifaciens and S.typhimurium), we assigned the catalytic domain (CD) sequence to belocated between amino acids 274-667 (SEQ ID NO:16). To clone thecatalytic domain of A. viscosus sialidase (AvCD), this region of the A.viscosus sialidase gene was engineered with codons optimized forexpression in E. coli (SEQ ID NO:15). The codon-optimized AvCDnucleotide sequence encoding amino acids 274-667 of the A. viscosussialidase (SEQ ID NO:15) was produced by chemical synthesis ofoverlapping oligonucleotides which were annealed, amplified by PCR andcloned into the expression vector pTrc99a (Amersham, N.J., USA).

Sialidase fusion constructs were made using standard molecular cloningmethods. The His₆-AvCD construct was made by fusing six histidines(His₆) to the N-terminal residue of the AvCD sequence. The His₆-AvCDconstruct has the nucleotide sequence of SEQ ID NO:28 and translatedamino acid sequence of SEQ ID NO:29. These sequences are depicted inFIG. 5.

To make the AR-AvCD construct, an anchoring domain was directly fusedwith the N-terminal residue of the AvCD sequence. The anchoring domain,referred to as AR, was derived from the GAG binding sequence of humanamphiregulin precursor (GenBank # AAH09799). Nucleotide sequencesencoding amino acids 125 to 145 (FIG. 2, SEQ ID NO:7) of the humanamphiregulin precursor were synthesized chemically as two overlappingoligonucleotides. The AR-AvCD construct has the nucleotide sequence ofSEQ ID NO:18 and translated amino acid sequence of SEQ ID NO:19.

Another construct, AR-G4S-AvCD, was made by fusing the same AR-encodingsequence used in the AR-AvCD construct with a sequence encoding afive-amino-acid linker (GGGGS; SEQ ID NO:10) which then was fused withthe AvCD sequence such that in a translation product, the linker wasfused to N-terminus of the catalytic domain of the A. viscosussialidase. The nucleotide sequence (SEQ ID NO:36) and translated aminoacid sequence (SEQ ID NO:37) of this construct are depicted in FIG. 7.All constructs were cloned into the pTrc99a expression vector.

In addition, four constructs were made in which the catalytic domain ofthe A. viscosus sialidase was fused to the N-terminus of the AR(GAG-binding domain of human amphiregulin; SEQ ID NO:7). In Construct #4(SEQ ID NO:21), the catalytic domain of the A. viscosus sialidaseconsisted of amino acids 274-666 of SEQ ID NO:12 fused to theGAG-binding domain of amphiregulin (SEQ ID NO:7). In Construct #5 (SEQID NO:23), the catalytic domain of the A. viscosus sialidase consistedof amino acids 274-681 of SEQ ID NO:12 fused to the GAG-binding domainof amphiregulin (SEQ ID NO:7). In Construct #6 (SEQ ID NO:25), thecatalytic domain of the A. viscosus sialidase consisted of amino acids290-666 of SEQ ID NO:12 fused to the GAG-binding domain of amphiregulin(SEQ ID NO:7). In Construct #7 (SEQ ID NO:27), the catalytic domain ofthe A. viscosus sialidase consisted of amino acids 290-681 of SEQ IDNO:12 fused to the GAG-binding domain of amphiregulin (SEQ ID NO:7). Allof these constructs displayed comparable sialidase activity in assays.

Example 9 Production of Sialidase Catalytic Domain Fusion Proteins

To produce the sialidase fusion proteins, the expression constructs weretransformed into E. coli BL21. A single colony was inoculated into 2.5ml of LB broth and grown overnight at 37° C. with shaking. In themorning 2 ml of overnight culture was inoculated into 500 ml of TBmedium in a 2 liter shake flask and the culture was allowed to grow toOD₆₀₀=4.0 (2-4 hours) at 37° C. with shaking. Protein expression wasinduced by addition of IPTG to a final concentration of 1 mM andcontinued for 3 hr with shaking Cells were harvested by centrifugationat 5,000×g for 10 min. Cell were washed once (resuspended in PBS andrecentrifuged) and resuspended in 15 ml of Lysis buffer. Compositions ofmedia and buffers used in protein expression and purification. TB mediumfor protein expression

Solution 1

-   -   Bacto-tryptone—12 g    -   Yeast extract—24 g    -   H₂O to 800 ml

Solution 2

-   -   KH₂PO₄ (anhydrous)—2.3 g    -   K₂HPO₄ (anhydrous)—12.5 g    -   H₂O to 100 ml

Autoclave solutions 1 and 2 separately, cool, mix and add the following:

-   -   60 ml of 20% glycerol (filter sterilized)    -   20 ml of 20% glucose (filter sterilized)

Lysis Buffer

50 mM phosphate, pH 8.0

10% glycerol

300 mM NaCl

Bacterial cells suspended in lysis buffer were lysed by sonication andcell debris was removed by centrifugation. Clarified lysate was passedthrough an SP-Sepharose column (bed volume 15 ml, flow rate 120cm/hour). The column was reconditioned to lower pH and salt with onevolume of PBS to ensure good retention of Fludase during endotoxinremoval. Endotoxin was removed by washing the column with 5 volumes ofPBS containing 1% Triton X-100, 0.5% Sodium Deoxycholate and 0.1% SDS.The detergents were washed away with 3 volumes of PBS and 3 volumes oflysis buffer. Proteins were eluted from the column with lysis bufferthat contained 0.8 M NaCl. The fraction eluted from SP-Sepharose wasadjusted to 1.9 M (NH₄)₂SO₄ (most contaminating proteins are salted outat this step) and clarified by centrifugation. The supernatant wasloaded onto Butyl-Sepharose column (flow rate 120 cm/hour). The columnwas washed with 2 volumes of 1.3 M (NH₄)₂SO₄ and the fusion was elutedwith 0.65 M (NH₄)₂SO₄. For the final step, size exclusion chromatographywas performed on Sephacryl S-200 equilibrated with PBS buffer at a flowrate of 25 cm/hour. Sialidase activity was determined against 4-MU-NANAas described in the following paragraph. Protein concentration wasdetermined using Bio-Rad's Bradford kit. Protein purity was assessed bySDS-PAGE and estimated to be >98%. Specific activity of the enzyme wasabout 937 U/mg. Endotoxin in final preparations was measured using LALtest (Cambrex) and estimated to be <0.5 EU/ml.

For purification of His6 containing fusion protein, cation exchange onSP-Sepharose was replaced with Metal Chelate Affinity Chromatography onNi-NTA. All buffers remained the same with the exception that elutionfrom Ni-NTA was performed by 0.25 M imidazole in lysis buffer.

Example 10 Sialidase Assay to Measure Activity of Sialidase CatalyticDomain Fusion Proteins

The sialidase activity of the AR-AvCD protein encoded by Construct #2was assayed and compared with that of native sialidases purified from C.perfringens (Sigma, St. Louis, Mo.) and A. ureafaciens (Prozyme, SanLeandro, Calif.). In addition, a fusion protein produced from aconstruct in which the amphiregulin GAG sequence (SEQ ID NO: 7) wasfused to the Neu 2 human sialidase (SEQ ID NO:8) was also assayed forsialidase activity.

The sialidase activity expressed as units per mg sialidase was measuredby the sialidase assay using the artificial fluorogenic substrate4-MU-NANA (Sigma). One unit of sialidase is defined as the amount ofenzyme that releases 10 nmol of MU from 4-MU-NANA in 10 min at 37° C.(50 mM CH₃COOH—NaOH buffer, pH 5.5) in reaction that contains 20 nmol of4-MU-NANA in a 0.2 ml volume. Reactions are stopped by addition of 1 mlof 0.2 M glycine/NaOH pH 10.2. Fluorescence emission is measured on afluorometer with excitation at 365 nm and emission at 445 nm, using4-methylumbelliferone (4-MU) to obtain a calibration curve (Potier etal., Anal. Biochem., (1979) 94 (2), 287-296).

TABLE 2 Specific activity of sialidases (units per mg). SialidaseSpecific activity AR-NEU2 8 AR-AvCD 937 C. perfringens 333 A.ureafaciens 82

Our results show that the AvCD fusion protein (AR-AvCD) has the highestspecific activity among all the tested sialidases (Table 2). Thespecific activity of AR-AvCD is over 100 times higher than that of ahuman sialidase fusion (AR-NEU2), and over two times higher than that ofC. perfringens sialidase. Experimental results comparing the stabilityof the sialidases indicate very high stability of AR-AvCD: No loss ofactivity for AR-AvCD was detected after 20 weeks at 25° C. or 4° C. insolution. By comparison, AR-NEU2 solution exhibited a half-life of 5 and2 weeks when stored at 25° C. and 37° C., respectively.

Example 11 Optimization of the N-Terminus of Sialidase Catalytic DomainFusion Proteins

The N-terminus of the AR-AvCD fusion protein was partially cleaved undercertain conditions that resulted in small degrees of proteinheterogeneity in the purified AR-AvCD prep. To solve this problem, wedesigned an approach to optimize the N-terminus of the sialidase fusionconstruct. A library containing AR-AvCD with random amino acids at theN-terminus was constructed as follows. AR-AvCD was amplified by PCRusing a primer pair in which the primer annealing on 5′-end of the genecontained a randomized sequence in positions corresponding to aminoacids 2 and 3. The nucleotide sequence of the primer and the encodedamino acid sequence are shown below.

(SEQ ID NO: 32) ttttcgtctcccatgvnnvnnaagcgcaaaaaaaaaggcggca(SEQ ID NO: 33) MetXxxXxxLysArgLysLysLysGlyGly

In SEQ ID NO:32, “n” stands for any nucleotide (a, c, g, or t) and “v”stands for nucleotides a, g or c. By designing the sequence in such away (disallowing the nucleotide t in the first position of codons) weavoided introduction of stop codons as well as aromatic amino acids(Phe, Tyr, Trp) and Cys. The Esp3I restriction endonuclease site (shownin bold) was introduced to allow generation of NcoI compatible overhang.The primer annealing to 3′-end of the gene carried HindIII sitefollowing the stop codon. The PCR product was digested withEsp3I-HindIII was ligated into pTrc99a expression vector digested withNcoI-HindIII. The ligation mix was transformed into E. coli and thecells were grown overnight in liquid culture containing Ampicillin.

The next day the culture was diluted with fresh medium, grown toOD₆₀₀=0.8 and induced with IPTG for 2 hours. Cells were harvested,homogenized and the fusions were subjected to two-step purification byliquid chromatography. Clarified lysate was loaded onto SP-Sepharoseequilibrated with lysis buffer (50 mM HEPES, pH 8.0, 0.3 M NaCl, 10%glycerol). The column was washed with 0.45 M NaCl and the fusions wereeluted with 0.9 M NaCl. The eluate was diluted with 10% glycerol tobring the concentration of NaCl to 0.2 M and loaded ontoHeparin-Sepharose column. The column was developed with a lineargradient of NaCl. The fractions that contained sialidase activity wereresolved on SDS-PAGE, electroblotted onto PVDF membrane and the 43 kDaband was subjected to amino-terminal sequencing.

The predominant N-terminal residues of the isolated sialidase fusionprotein were either Val or Gly followed by the N-terminal residues ofthe AR tag. We then synthesized new sialidase fusion constructs,Constructs #2 and #3, by introducing a Val in front of the AR sequencesuch that the first six amino acids encoded by Constructs #2 and #3 were(Met-Val-Lys-Arg-Lys-Lys (SEQ ID NO:17)). N-terminal sequencing ofproteins made from these new fusion constructs showed 100% homogeneitywith the initiation Met being completely removed (which is desirable fortherapeutic proteins) and Val being the first N-terminal residuefollowed by the AR tag sequence. These data are consistent with earlierpublications that reported the common rules of N-terminal processing andprotein stability as function of protein's N-terminal amino acid residue(Hirel et al., Proc. Natl. Acad. Sci. U.S. A, (1989) 86 (21), 8247-8251;Varshaysky, Proc. Natl. Acad. Sci. U.S. A, (1996) 93 (22), 12142-12149).

The nucleotide sequences of new fusion Construct #2 (AR-AvCD withoptimized N-terminus) (SEQ ID NO:18) and its amino acid sequencetranslation (SEQ ID NO:19) is depicted in FIG. 6. The nucleotidesequences of new fusion Construct #3 (AR-G4S-AvCD with optimizedN-terminus) (SEQ ID NO:36) and its amino acid sequence translation (SEQID NO:37) is depicted in FIG. 7. The amino acid sequence of processedproteins isolated from E. coli infected with Construct #2 is providedherein as SEQ ID NO:38 and the amino acid sequence of processed proteinsisolated from E. coli infected with Construct #3 is provided herein asSEQ ID NO:39.

Example 12 Comparing Activities of Sialidase Constructs with or withoutan Anchoring Domain

To evaluate if the AR sequence indeed improves the cell-surface activityof a sialidase fusion protein, we incubated proteins purified from E.coli that were transformed with Construct #2; SEQ ID NO:18, depicted inFIG. 6) or Construct #1 (His₆-AvCD; SEQ ID NO:28, depicted in FIG. 5)with primary human bronchial epithelial cells and measured cell-boundsialidase activity after extensive washing. For cells incubated withConstruct #2 protein (SEQ ID NO:19), up to 10% of the sialidase wasfound to be cell-bound, and the cell-bound sialidase activity increasedin a dose-dependent manner with the input concentration of Construct #2protein. However, Construct #1 protein (SEQ ID NO:29) incubated cellsonly exhibited background level of sialidase activity. Furthermore, wetreated MDCK cells with either Construct #2 protein or Construct #1protein and measured the level of residual α(2,6)-linked sialic acid onthe surface of the cells (FIG. 8). At equal levels of enzymatic activitybelow 100 mU per well, Construct #2 protein demonstrated significantlyhigher potency than Construct #1 protein. These results indicate thatthe AR domain indeed enhances the function of sialidase.

Example 13 In Vitro Activities of Sialidase Fusion Proteins Stocks ofInfluenza Viruses

Influenza viral strains are obtained from ATCC and the repository at St.Jude Children's Research Hospital. All experiments involving influenzaviruses are conducted at Bio-safety level II.

Viruses are propagated on Madin-Darby canine kidney (MDCK) cells inminimal essential medium (MEM) supplemented with 0.3% bovine serumalbumin and 0.5 micrograms of trypsin per ml. After incubating for 48 to72 hours, the culture medium is clarified by low speed centrifugation.Viral particles are pelleted by ultracentrifugation through a 25%sucrose cushion. Purified viruses are suspended in 50% glycerol-0.1MTris buffer (pH 7.3) and stored at −20° C.

1. Cell Protection Assay

To evaluate the ability of the Construct #2 AR-AvCD protein to protectcells against influenza viruses, we first treated MDCK cells withAR-AvCD made from Construct #2 or a broad-spectrum bacterial sialidaseisolated from A. ureafaciens, and challenged the cells with a broadselection of human influenza viruses (IFV), including human IFV A of H1,H2 and H3 subtypes, human IFV B as well as an avian IFV strain. As shownin FIG. 9, the fusion protein made from Construct #2 demonstrated 80 to100% of cell protection that was comparable to the effect of A.ureafaciens sialidase.

To perform the assay, MDCK cells were treated with 10 mU of AR-AvCDprotein (made using Construct #2) or the isolated sialidase of A.ureafaciens at 37° C. for 2 hrs. The cells were subsequently challengedwith influenza viruses at MOI 0.1 for 1 hr. The cells were washed andincubated in fresh DMDM:F12 supplemented with 0.2% ITS (GIBCO) and 0.6μg/ml acetylated trypsin (Sigma). The cells were stained with 0.5%crystal violet and 20% methanol for 5 min and rinsed with tap water. Thelevel of viable cells in each well was quantitated by extracting crystalviolet by 70% ethanol and reading at 570 nM. Cell protection wascalculated by 100×{(sialidase treated sample−virus only)/(uninfectedsample-virus only)}.

IFV Inhibition Assay

We evaluated inhibition of IFV amplification by AR-AvCD protein (madeusing Construct #2) and AR-G₄S-AvCD protein (made using Construct #3)using a cell-based ELISA method (Belshe et al., J. Virol., (1988) 62(5), 1508-1512).

To perform the assay, MDCK monolayers in 96 well plates were treatedwith 16 mU of the sialidases AR-AvCD made from Construct #2 orAR-G₄S-AvCD made from Construct #3 in EDB/BSA buffer (10 mM SodiumAcetate, 150 mM NaCl, 10 mM CaCl₂, 0.5 mM MgCl₂, and 0.5% BSA) for 2 hrsat 37° C. Both the sialidase treated and the untreated control cells(treated with only EDB/BSA buffer) were infected with 0.1 MOI of virus.After 1 hour, the cells were washed two times with PBS and incubated inDMEM:F12 supplemented with 0.2% ITS (Gibco) and 0.6 ug/ml acetylatedtrypsin (Sigma). Forty to 48 hours post-infection, the levels ofcell-bound virus were determined by using a cell-based ELISA assay.Specifically, cells were fixed in 0.05% glutaraldehyde in PBS and wereincubated with 50 μl of 10³ dilution of either anti-influenza A NPantiserum or anti-influenza B (Fitzgerald Inc.) in 0.5% BSA and PBS at37° C. for 1 hr. After washing, each well was incubated with HRP-proteinG in 0.5% BSA and PBS for 1 hr. After final washes, 50 μl of 25 mMsodium citrate (pH 4.5) containing 0.02% 3,3′,5,5′-tetramethylbenzidinedihydrochloride (Sigma) and 0.01% hydrogen peroxide was allowed to reactwith the cells at room temperature for 5 min. The reactions were stoppedby adding 50 μl of 1M H₂SO₄, and quantitated by measuring opticaldensities at 450 nM. Percentage viral replication inhibition iscalculated by 100%×{(virus only samples−sialidase treatedsamples)/(virus only samples−uninfected samples)}.

Data on inhibition of viral replication and cell protection EC50's andselective indexes for recombinant sialidase fusion proteins AR-AvCD madefrom Construct #2 and AR-G₄S-AvCD made from Construct #3 for a varietyof human influenza A and influenza B viruses, as well as equine virusesare shown in FIG. 12.

As shown in FIG. 10, sialidase fusion proteins strongly inhibitedamplification of a broad selection of influenza viruses. Notably,80-100% viral inhibition (FIG. 10) as well as cell protection (FIG. 9)was achieved although a maximum of 70-80% of cell surface sialic acidwas removed by the sialidase treatment (FIG. 8). This findingdemonstrates that it is unnecessary to completely eliminate cell surfacesialic acid in order to achieve the desired therapeutic effect oftreating with the sialidase fusion proteins of the present invention.The residual 20-30% of the surface sialic acid, while being inaccessiblefor the sialidase fusion proteins, is probably inaccessible forinfluenza viruses as well.

Cytotoxicity of Sialidase Fusion Proteins

To evaluate the cytotoxicity of AR-AvCD or AR-G₄S-AvCD proteins (madefrom Constructs #2 and #3), MDCK cells were seeded at low density in96-well plates and cultured for 5 days in DMEM containing 10% FBS and upto 20 U of AR-AvCD protein or AR-G₄S-AvCD protein per well (bothsialidases remained fully active during the entire experiment). Celldensity in AR-AvCD or AR-G₄S-AvCD treated or control wells weredetermined every day by staining the cells with crystal violet andmeasuring absorption at 570 nM. No inhibition of cell growth wasobserved even at the highest concentration of AR-AvCD or AR-G₄S-AvCD(100 U/ml) in the culture. Therefore IC₅₀, which is the drugconcentration that inhibits cell growth by 50%, for AR-AvCD orAR-G₄S-AvCD is above 100 U/ml.

Example 14 In Vivo Activities of Sialidase Catalytic Domain FusionProtein

Ferrets can be infected with human unadapted influenza viruses andproduce signs of disease comparable to those of humans, which can betreated by antiviral compounds, such as zanamivir (Relenza). (Mendel etal., Antimicrob Agents Chemother, (1998) 42 (3), 640-646; Smith andSweet, Rev. Infect. Dis., (1988) 10 (1), 56-75; Reuman et al., J. Virol.Methods, (1989) 24 (1-2), 27-34). To evaluate in vivo efficacy of ourcompounds, we tested AR-AvCD protein (made from Construct #2) in theferret model. Specifically, 24 young female ferrets (0.5-0.8 kg)(Marshall Farms, North Rose, N.Y.) that tested negative for the presenceof anti-hemagglutinin antibodies in sera were included in the study. Twoanimals were placed in each cage and allowed to acclimate for 3 daysbefore the experiment. The animals were randomly divided into threegroups: 8 animals were treated with drug dilution buffer and viralchallenge, 12 animals were treated with AR-AvCD and viral challenge, and4 animals were treated with AR-AvCD only. A preparation of AR-AvCDdissolved in phosphate buffered saline (PBS) that contains 500 U/ml insialidase activity and 0.7 mg/ml in protein concentration was used inthe study. Animals in the drug treatment groups received 1 ml of AR-AvCDat each dose, which amounts to about 1 mg/kg in dosage level.

Ferrets were anesthetized and inoculated intranasally (0.5 ml into eachnostril) with AR-AvCD or PBS twice (8 am and 6 pm) and daily for a totalof 7 days (2 days prior to the viral challenge and 5 days post virusinoculation). The ferrets were observed following the drug applicationfor signs of intolerance. Viral inoculation was carried out on day 3between 10-11 am. The viral challenge was done with human A/Bayern/7/95(H1N1)-like virus at dose 10⁵ TCID₅₀ (≧10⁴ ferret ID₅₀). The nasalwashes were collected from all animals starting day 2 post AR-AvCDtreatment and continued until day 7. To collect nasal washes, 1 ml ofsterile PBS was administered intranasally, the sneezed liquid washarvested and its volume was recorded. The nasal washes werecentrifuged. The pelleted cells were re-suspended and counted in ahemacytometer under a microscope. The supernatants were collected,aliquoted and stored at −80° C. The protein concentration in cell-freenasal washes was determined by using the Bio-Rad protein reagentaccording to manufacturer's protocol (Bio-Rad, Hercules, Calif.). Forvirus titration of the nasal washes, inoculated MDCK cells wereincubated for 3 days at 36° C. in a CO₂ incubator. The monolayers wereinspected visually for cytopathic effect (CPE) and aliquots of the cellculture supernatants from each well were tested for the virus presenceby a standard hemagglutination assay with guinea pig red blood cells.Viral titer was determined by the Spearman Karber method ((1996)).

In uninfected animals given intranasal AR-AvCD (n=4), no apparent effecton the inflammatory cell counts and protein concentrations in the nasalwashes was observed (FIGS. 15 A and B). Nasal washes from these animalswere followed for 7 days and were all negative for viral shedding. Nosigns of drug-related toxicity were detected in these animals at thedrug dose used in this study. In the vehicle-treated group, virusreplicated in the nasal epithelium of all 8 ferrets. Viral sheddingreached peak values of 4.4 to 5.9 log₁₀TCID₅₀ (mean peak titer of 4.9)on day 1 or 2 post challenge, diminished over time and became negativeby day 5 (FIG. 13). By contrast, only 3 of 12 AR-AvCD-treated ferretswere positive for viral shedding on day 1 post challenge (FIG. 13), andtheir nasal viral titers were about 100 times lower than those in thevehicle-treated animals (mean 2.4±0.3 vs. 4.4±0.4 log₁₀TCID₅₀) (FIG.13). After day 1, the response to the AR-AvCD treatment variedsubstantially. Three animals were completely protected againstinfection, signs of illness, and inflammatory response (FIG. 13), ferrettag #803, 805, 806). The protection was also confirmed by a lack ofseroconversion on day-14 post challenge. One ferret (tag #780) did notshed virus during the first three days post challenge, but it died onday 4 post infection from an unrelated injury. The shedding in theremaining 8 ferrets varied during the course of infection, ranging fromferret #812 that shed virus for a day only, to the ferret #791 that shedvirus for 5 days.

Infection in the ferrets that shed virus for at least one day wasconfirmed by more than a 16-fold rise in the post-challenge anti-HAantibody titer (seroconversion). There was no apparent effect of AR-AvCDtreatment on the anti-HA titers in post-challenge sera (320-1280, vs.160-1280, vehicle- and drug-treated group, respectively).

In ferrets that shed the virus despite the AR-AvCD treatment (n=8), theinflammatory response was reduced and animals appeared to be more alertand active compared to the untreated ferrets that were invariablylethargic and feverish. For this group of 8 infected, AR-AvCD-treatedanimals, the mean AUC (area under the curve) value calculated for thenasal protein concentrations was reduced by approximately 40% (2.68 vs.4.48, arbitrary units) compared to the vehicle-treated infected animals(FIG. 11B). In vehicle-treated infected animals, the number ofinflammatory cells in nasal washes was increased to approximately100-fold above those in uninfected animals on day 2 post challenge.These levels were sustained for 4 additional days. The AR-AvCD-treatedanimals exhibited a significant reduction in the number of inflammatorycells in the nasal washes. Specifically, the AUC value for cell countswas reduced by approximately 3-fold in the AR-AvCD-treated animalscompared to the vehicle-treated infected animals (1965 vs. 674,arbitrary units, FIG. 11A). The observed reduction in the inflammatoryresponse indicates the importance of inhibiting viral replication at theearly stage of infection.

Example 15 Inhibition of Bacterial Cell Adhesion by Sialidase FusionProteins Bacteria

S. pneumoniae: 10 encapsulated strains of different serotypes areselected from the clinical isolates deposited at ATCC. Bacteria aremaintained as frozen stocks and passaged on tryptic soy agar platescontaining 3% sheep blood (Difco & Micropure Medical Inc.) for 18 hr at37° C. in 5% CO₂. To label pneumococci with radioisotope, an inoculum istaken from a 1- to 2-day plate culture, added to lysine-deficienttryptic soy broth containing 70 μCi of [³H] lysine per ml and incubatedat 37° C. in 5% CO₂. The growth of each culture is monitored by lightabsorbance at 595 nm. At late logarithmic phase, the bacteria areharvested, washed twice by centrifugation (13,000 rpm×3 min), andresuspended in L-15 medium (without phenol red) plus 0.1% BSA (L-15-BSA)(Cundell and Tuomanen, Microb. Pathog., (1994) 17 (6), 361-374)(Barthelson et al., Infect. Immun., (1998) 66 (4), 1439-1444).

H. influenzae: 5 strains of type b (Hib) and 10 nontypable strains(NTHi) are obtained from the clinical isolates deposited at ATCC. Allstrains are stocked in brain heart infusion (BHI, Difco) containinghemin (ICN) and NAD (Sigma) and kept frozen until use; then they arecultured on BHI agar supplemented with hemin and NAD and grown for 14 hrat 37° C. with 5% CO₂. (Kawakami et al., Microbiol. Immunol., (1998) 42(10), 697-702). To label the bacteria with [³H], H. influenzae cells areinoculated in BHI broth containing hemin, NAD and [³H]leucine at 250μCi/ml and allowed to grow until late logarithmic phase and thenharvested, washed and resuspended in L-15-BSA (Barthelson et al.,Infect. Immun., (1998) 66 (4), 1439-1444).

Cell Adhesion Assay

All [³H]-labeled bacteria are suspended in L-15-BSA after washing, thebacterial concentration is determined by visual counting with aPetroff-Hausser chamber, radioactivity is determined by scintillationcounting, and the specific activity of the [³H]-labeled cells iscalculated. Preparations of bacteria with 7 cpm/1000 cells or greaterare used. The bacteria are diluted to 5×10⁸ cells/ml. BEAS-2B cellmonolayers are incubated with [³H]-labeled bacterial suspensioncontaining 5×10⁷ bacteria at 37° C. in 5% CO₂. After 30 min, unboundbacteria are removed by washing with L-15-BSA for 5 times. Bacteriaattached to the WD-HAE tissue samples are quantitated by scintillationcounting.

Desialylation of BEAS-2B Cells by Sialidase Fusion Proteins and Effectson Cell Adhesion By H. influenzae and S. pneumoniae.

BEAS-2B cells are incubated with 1-50 mU of AR-AvCD for 2 hours. Celladhesion assay will be carried out using H. influenzae and S. pneumoniaestrains as described above. Mock treated cells are used as positivecontrol. Efficacy of AR-AvCD is quantitated as the EC₅₀, which is theamount of enzyme to achieve 50% inhibition on bacterial adhesion.

Example 16 Improving Transduction Efficiency of AAV Vector UsingSialidase Fusion Proteins In Vitro Experiments

An experiment demonstrating effect of AR-AvCD is performed in a waysimilar to the procedure published (Bals et al., J. Virol., (1999) 73(7), 6085-6088). A monolayer of Well-Differentiated Airway Epithelium(WDAE) cells is maintained in transwells (Karp et al., Methods Mol.Biol., (2002) 188, 115-137; Wang et al., J. Virol., (1998) 72(12),9818-9826). In order to eliminate sialic acid from the cell surface theculture medium is replaced with serum free medium in which 0.5-10 unitsof AR-AvCD are dissolved. The cells are treated for 30 min to 6 hours.The cell monolayers are washed, transduced with AAV, and transductionefficiency is estimated using standard procedures. Several transwellsare treated with medium only (without AR-AvCD) to serve the purpose ofcontrol (basal transduction efficiency). Additional controls may includethe transwells treated with AR-AvCD only to assess cytotoxic effect ofdesialylation. A reporter virus is used for facile detection oftransduced cells. Examples of reporter AAV and their use have beendescribed in literature and include AAV-CMV-eGFP, AAV2LacZ (Bals et al.,J. Virol., (1999) 73 (7), 6085-6088; Wang et al., Hum. Gene Ther.,(2004) 15 (4), 405-413) and alkaline phosphatase (Halbert et al., Nat.Biotechnol., (2002) 20 (7), 697-701). The efficiency is estimated bylight microscopy of the cells that were fixed and treated withappropriate substrate (if lacZ or AP containing virus is used) orfluorescent microscopy of live cells (if GFP is used). According to theexperiments conducted at NexBio with NHBE primary epithelial cells(Cambrex, Walkersville, Md.) the maximum amount of removal of sialicacid is achieved in less than one hour when 10 units of AR-AvCD pertranswell are used. Other cell lines used (e.g. MDCK) becomedesialylated with much less AR-AvCD administered (0.1 U for 1 hour). Itis therefore our estimate that a treatment of WDAE with 10 U of AR-AvCDfor 2 hours will be sufficient to remove accessible sialic acid andprovide significant enhancement of transduction of WDAE cells with AAV.

Testing Effect of AR-AvCD Treatment on AAV Transduction in an AnimalModel.

To demonstrate effect of AR-AvCD treatment in animal model an experimentsimilar to previously described is conducted (Flotte et al., Proc. Natl.Acad. Sci. U.S. A, (1993) 90 (22), 10613-10617; Halbert et al., Nat.Biotechnol., (2002) 20 (7), 697-701). Several hours (1-6) prior toadministration of AAV AR-AvCD is delivered to mice lungs by nasalaspiration of aerosol or lyophilized AR-AvCD powder according topreviously published protocol (Flotte et al., Proc. Natl. Acad. Sci.U.S. A, (1993) 90 (22), 10613-10617). AAV carrying reporter gene(alkaline phosphatase) is delivered by nasal aspiration, mice areeuthanized 4 weeks later and transduced cells are detected in fixedlungs as previously described (Halbert et al., J. Virol., (1998) 72(12), 9795-9805).

Example 17 Sialidase Treatment Inhibits Mast Cell Functions and SmoothMuscle Contraction in the Trachea

Using experimental methods described previously (Cocchiara et al., J.Neuroimmunol., (1997) 75 (1-2), 9-18), it will be demonstrated thattreatment by compounds of the present invention prevents substance P(SP)induced histamine release by mast cells. Using another set ofexperiments (Stenton et al., J. Pharmacol. Exp. Ther., (2002) 302(2),466-474), treatment by compounds of the present invention will inhibitβ-hexosaminidase release by mast cells stimulated by two PAR-activatingpeptides (PAR stands for proteinase-activated receptors).

Compounds of the present invention will be administered intratracheallyin guinea pigs and the airway reactivity will be assessed in the animalsas described previously (Jarreau et al., Am. Rev. Respir. Dis., (1992)145 (4 Pt 1), 906-910; Stenton et al., J Pharmacol. Exp. Ther., (2002)302(2), 466-474). Sialidase treatment should not induce nonspecificairway hyperreactivity judged by the reaction to multiple inducers. Inaddition, sialidase treatment should reduce substance P-inducedbronchoconstriction. Similarly, compounds of the present invention willbe used to treated the isolated guinea pig and rat trachea and lung (Kaiet al., Eur. J. Pharmacol., (1992) 220 (2-3), 181-185; Stenton et al.,J. Pharmacol. Exp. Ther., (2002) 302(2), 466-474). Again recombinantsialidase treatment will have no effect on smooth muscle contractionsinduced by acetylcholine, histamine and 5-hydroxytryptamine. Inaddition, it will inhibit tracheal contraction induced by antigen(ovalbumin) or compound 48/80.

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All publications, including patent documents, Genbank sequence databaseentries including nucleotide and amino acid sequences and accompanyinginformation, and scientific articles, referred to in this applicationand the bibliography and attachments are incorporated by reference intheir entirety for all purposes to the same extent as if each individualpublication were individually incorporated by reference.

All headings are for the convenience of the reader and should not beused to limit the meaning of the text that follows the heading, unlessso specified.

1. A fusion protein, comprising at least one catalytic domain of asialidase and at least one anchoring domain, wherein the catalyticdomain sequence comprises: a) the sequence of amino acids extending fromamino acid 274 to amino acid 681 of SEQ ID NO:12, inclusive and lacksthe sequence that consists of the sequence extending from amino acid 1to amino acid 273 and from amino acid 682 to amino acid 901 of SEQ IDNO:12; b) the sequence of amino acids extending from amino acid 290 toamino acid 666 of SEQ ID NO:12, inclusive and lacks the sequence thatconsists of the sequence extending from amino acid 1 to amino acid 289and from amino acid 667 to amino acid 901 of SEQ ID NO:12; or c) thesequence of amino acids extending from amino acid 290 to amino acid 681of SEQ ID NO:12, inclusive and lacks the sequence that consists of thesequence extending from amino acid 1 to amino acid 289 and from aminoacid 682 to amino acid 901 of SEQ ID NO:12; and the anchoring domaincomprises the GAG-binding domain of human platelet factor 4 (SEQ IDNO:2), the GAG-binding domain of human interleukin 8 (SEQ ID NO:3), theGAG-binding domain of human antithrombin III (SEQ ID NO:4), theGAG-binding domain of human apoprotein E (SEQ ID NO:5), the GAG-bindingdomain of human angio-associated migratory protein (SEQ ID NO:6), or theGAG-binding domain of human amphiregulin (SEQ ID NO:7). 2.-28.(canceled)